High-throughput screening of expressed DNA libraries in filamentous fungi

ABSTRACT

The invention provides a method for the expression of exogenous DNA libraries in filamentous fungi. The fungi are capable of processing intron-containing eukaryotic genes, and also can carry out post-translational processing steps such as glyclosylation and protein folding. The invention provides for the use of fungi with altered morphology, which permits high-throughput screening and directed molecular evolution of expressed proteins. The same transformed fungi may be used to produce larger quantities of protein for isolation, characterization, and application testing, and may be suitable for commercial production of the protein as well.

SUMMARY OF THE INVENTION

[0001] The invention provides a method for the expression and subsequentscreening of DNA libraries, particularly synthetic, genomic, and cDNAlibraries, in filamentous fungal hosts. The system employs transformedor transfected filamentous fungal strains which generate transferablereproductive elements, for example by efficient sporulation, insubmerged culture. The fungi preferably exhibit a morphology thatminimizes or eliminates the formation of entangled mycelia. Particularlypreferred fungal strains are also capable of expressing isolatablequantities of exogenous proteins for evaluation. The mutant fungalstrains of the invention are particularly well-suited forhigh-throughput screening techniques, due to their production oftransferable reproductive elements, high levels of expression, and verylow culture viscosity.

BACKGROUND OF THE INVENTION

[0002] Naturally-occurring populations of microorganisms exhibit a widearray of biochemical and metabolic diversity. Due in part todifficulties in isolating and culturing many microorganisms, a vastnumber of potentially valuable proteins and polypeptides present inthese populations have escaped identification. Indeed, it has beenestimated that less than one percent of the world's microorganisms havebeen cultured to date. There remains a pressing need for new approachesto the characterization of proteins, polypeptides and metabolites fromas-yet uncultivated, unidentified microorganisms, and also from knownmicroorganisms. (The term “protein” as used hereinafter should beunderstood to encompass peptides and polypeptides as well.) There alsoremains a need for new approaches to the identification and isolation ofthe genes encoding these proteins, so as to enable the modificationand/or production of the proteins.

[0003] One approach to this problem has been described by Short in U.S.Pat. Nos. 5,958,672; 6,001,574, 6,030,779, and 6,057,103 (the contentsof which are incorporated herein by reference). In this approach, agenomic DNA library is prepared directly from an environmental sample(e.g. a soil sample), with or without making an attempt to isolate orculture any organisms that might be present. The DNA library isexpressed in E. coli, and the expressed proteins are screened for aproperty or activity of interest. Short alludes to, but does notdescribe or enable, the use of fungal host cells in this method.

[0004] The approach as described suffers from several seriousdisadvantages, one of which is that E. Coli does not effectively expressgenes having introns. Roughly 90% of the species of microorganisms insoil are eukaryotes (principally fungi), which generally do have intronsin their genomic DNA. Given that there are already about 100,000 speciesof eumycotan fungi known, with an estimated 1,000,000 yet to bediscovered (B. Kendrick, The Fifth Kingdom, Mycologue Publications1999), the potential for protein and metabolite diversity is far higheramong the fungal genomes, but the presence of introns puts most of thefungal protein and metabolite repertoire out of the reach of bacterialexpression systems. Not only are many classes of enzymes (e.g.,secretory fungal lignin peroxidases and manganese-dependent peroxidases)unique to fungi, but there are many fungal proteins, including enzymes(e.g. lignin peroxidases, A. niger invertase), that are glycosylated,and such proteins would not be glycosylated if expressed by E. coli. Themuch higher number and greater size and complexity of fungal genomes,the uniqueness of many fungal proteins, and the glycosylation of manyfungal proteins, all indicate that the fraction of microbial protein andmetabolite diversity in a given environmental sample that could beactually detected by bacterial expression of genomic DNA is considerablyless than 10%.

[0005] Due in part to the spread of AIDS and the rising population oforgan transplant recipients, there is a growing population ofimmune-compromised or immuno-supressed individuals, and the number andvariety of fungal infections has grown apace (Infect. Med. 16:380-382,385-386 (1999)). There is a need to identify and characterize proteinsfrom pathogenic fungi in the ongoing search for new targets foranti-fungal drugs, which requires the capability to screen DNA librariesderived from fungal genomes. Again, the presence of introns in fungalgenomes makes expression of genomic DNA libraries difficult in mostcurrently available bacterial hosts. There has also been a rise in theprevalence of antibiotic-resistant bacterial infections, creating a needfor high-throughput screening for new fungal metabolites havingantibiotic activity.

[0006] Eukaryotic genomes of higher organisms are also too complex forcomprehensive expression of DNA libraries in bacteria. When alleukaryotic species are considered, bacteria represent only about 0.3% ofall known species (E. O. Wilson, “The Current State of BiologicalDiversity”, in Biodiversity, National Academy Press, Washington D.C.,1988, Chapter 1); thus the fraction of the world's genetic diversityaccessible to bacterial expression systems is extremely limited.

[0007] To avoid problems with introns, it is possible to prepare a cDNAlibrary and express it in bacteria. However, this approach relies uponthe presence of RNA transcripts, and any genes not actively beingtranscribed will not be represented in the library. Many desirableproteins are expressed only under specific conditions (e.g., virulencefactors in pathogenic fungi) and these conditions may not exist at thetime the mRNA is harvested. Furthermore, in order to obtain sufficientRNA to prepare a cDNA library, it is necessary to culture a fair amountof the organism. For organisms in environmental samples that do not growwell in culture, or novel microorganisms for which appropriate cultureconditions are unknown, sufficient RNA will not be readily or reliablyobtained. In contrast, sufficient genomic DNA can be obtained from avery small number of individual cells by PCR amplification, using eitherrandom primers or primers designed to favor certain classes of genes.Finally, genes that are highly expressed in an organism will tend to beover-represented in the mRNA, and thus over-represented at the expenseof minimally-expressed genes in a cDNA library. In order to have a highlevel of coverage of the mRNA species present, a much larger number ofclones must be screened if a cDNA library is employed instead of agenomic library, since the latter will have a more nearly equalrepresentation of the variety of genes present. Clearly it is moredesirable to screen a genomic DNA library if at all possible.

[0008] Also, E. coli is incapable of secretion of many proteins, andthus is undesirable as a host cell for screening purposes where thescreening relies upon secretion of the gene product. An additionaldisadvantage for E. coli, and for bacterial hosts in general, is thatprokaryotes cannot provide many of the post-translational modificationsrequired for the activity of numerous eukaryotic proteins. In additionto glycosylation, subunit cleavage, disulfide bond formation, and properfolding of proteins are examples of the post-translational processingoften required to produce an active protein.

[0009] To ensure such processing one can sometimes use mammalian cells,but mammalian cells are difficult to maintain, require expensive media,and are not generally transformed with high efficiency. Suchtransformation systems are therefore not convenient for high-throughputscreening of proteins, although efforts have been made to employmammalian cells as hosts for cDNA library screening (Schouten et al., WO99/64582). An approach involving fusion of transformed protoplasts withmammalian cells prior to library screening has been described (U.S. Pat.No. 5,989,814), but expression of the protein library occurs in bacteriaor yeast prior to cell fusion. There have been efforts to modifyglycosylation patterns enzymatically after expression in host cells(Meynial-Salles and Combes, J. Biotechnol., 46:1-14 (1996)), but suchmethods must be tailored for specific products and are not suitable forexpression of proteins from a DNA library. More recently, Maras et al.,Eur. J. Biochem., 249:701-707 (1997) (see also U.S. Pat. No. 5,834,251)have described a strain of Trichoderma reesei engineered to expresshuman GlcNAc transferase I. The enzyme transfers N-acetylglucosainine tomannose residues on other expressed exogenous proteins, a first steptoward more closely approximating natural mammalian products.

[0010] The use of yeast as host cells solves some of the above problems,but introduces others. Yeast tend to hyper-glycosylate exogenousproteins (Bretthauer and Castellino, 1999, Biotechnol. Appl. Biochem.30:193-200), and the altered glycosylation patterns often renderexpressed mammalian proteins highly antigenic (C. Ballou, in MolecularBiology of the Yeast Sacccharomyces, J. Strathern et al., eds., ColdSpring Harbor Laboratory Press, NY, 1982, 335-360). Although yeast arecapable of coping with a limited number of introns, they are notgenerally capable of handling complex genes from higher species such asvertebrates. Even genes from filamentous fungi are usually too complexfor yeast to transcribe efficiently, and this problem is compounded bydifferences in expression and splicing sequences between yeast andfilamentous fungi (see e.g., M. Innis et al., Science 1985 228:21-26).Despite these drawbacks, transformation and expression systems for yeasthave been extensively developed, generally for use with cDNA libraries.Yeast expression systems have been developed which are used to screenfor naturally secreted and membrane proteins of mammalian origin (Klein,et al, Proc. Natl. Acad. Sci. USA 1996 93:7108-7113; Treco, U.S. Pat.No. 5,783,385), and for heterologous fungal proteins (Dalboge andHeldt-Hansen, Mol. Gen. Genet. 243:253-260 (1994)) and mammalianproteins (Tekamp-Olson and Meryweather, U.S. Pat. No. 6,017,731).

[0011] The term “yeast” as used in the context of yeast expressionsystems generally refers to organisms of the order Saccharomycetales,such as S. cerevisiae and Pichia pastoris. For the purposes of thisdisclosure, the terms “fungi” and “fungal” should be understood to referto Basidiomycetes, Zygomycetes, Oomycetes, and Chythridiomycetes, andAscomycetes of the class Euascomycetes, which are not of the orderSaccharomycetales. Filamentous fungi may be distinguished from yeast bytheir hyphal elongation during vegetative growth, and obligately aerobiccarbon catabolism (vegetative growth in yeast is accomplished by buddingfrom a unicellular thallus, and yeast may employ fermentativecatabolism.)

[0012] Proper intron splicing, and glycosylation, folding, and otherpost-translational modifications of fungal gene products would be mostefficiently handled by a fungal host species, making filamentous fungisuperior hosts for screening genomic DNA from soil samples. It alsomakes them excellent hosts for the production of fungal enzymes ofcommercial interest, such as proteases, cellulases, and amylases. It hasalso been found that filamentous fungi are capable of transcribing,translating, processing, and secreting the products of other eukaryoticgenes, including mammalian genes. The latter property makes filamentousfungi attractive hosts for the production of proteins of biomedicalinterest. Glycosylation patterns introduced by filamentous fungi moreclosely resemble those of mammalian proteins than do the patternsintroduced by yeast. For these reasons, a great deal of effort has beenexpended on the development of fungal host systems for expression ofheterologous proteins, and a number of fungal expression systems havebeen developed. For reviews of work in this area, see Maras et al.,Glycoconjugate J, 16:99-107 (1999); Peberdy, Acta Microbiol. Immunol.Hung. 46:165-174 (1999); Kruszewsa, Acta Biochim. Pol. 46:181-195(1999); Archer et al., Crit. Rev. Biotechnol. 17:273-306 (1997); andJeenes et al., Biotech. Genet. Eng. Rev. 9:327-367 (1991).

[0013] High-throughput expression and assaying of DNA libraries derivedfrom fungal genomes would also be of use in assigning functions to themany mammalian genes that are currently of unknown function. Forexample, once a fungal protein having a property of activity of interestis identified, the sequence of the encoding gene may be compared to thehuman genome sequence to look for homologous genes.

[0014] Yelton et al., U.S. Pat. No. 4,816,405, discloses themodification of filamentous Ascomycetes to produce and secreteheterologous proteins. Buxton et al., in U.S. Pat. No. 4,885,249, and inBuxton and Radford, Mol. Gen. Genet. 196:339-344 (1984), discloses thetransformation of Aspergillus niger by a DNA vector that contains aselectable marker capable of being incorporated into the host cells.McKnight et al., U.S. Pat. No. 4,935,349, and Boel, in U.S. Pat. No.5,536,661, disclose methods for expressing eukaryotic genes inAspergillus involving promoters capable of directing the expression ofheterologous genes in Aspergillus and other filamentous fungi. Royer etal., in U.S. Pat. No. 5,837,847, and Berka et al., in WO 00/56900,disclose expression systems for use in Fusarium venenatum employingnatural and mutant Fusarium spp. promoters. Conneely et al., in U.S.Pat. No. 5,955,316, disclose plasmid constructs suitable for theexpression and production of lactoferrin in Aspergillus. Cladosporiumglucose oxidase had been expressed in Aspergillus (U.S. Pat. No.5,879,921).

[0015] Similar techniques have been used in Neurospora. Lambowitz, inU.S. Pat. No. 4,486,533, discloses an autonomously replicating DNAvector for filamentous fungi and its use for the introduction andexpression of heterologous genes in Neurospora. Stuart et al. describeco-transformation of Neurospora crassa spheroplasts with mammalian genesand endogenous transcriptional regulatory elements in U.S. Pat. No.5,695,965, and an improved strain of Neurospora having reduced levels ofextracellular protease in U.S. Pat. No. 5,776,730. Vectors fortransformation of Neurospora are disclosed in U.S. Pat. No. 5,834,191.Takagi et al. describe a transformation system for Rhizopus in U.S. Pat.No. 5,436,158. Sisniega-Barroso et al. describe a transformation systemfor filamentous fungi in WO 99/51756, which employs promoters of theglutamate dehydrogenase genes from Aspergillus awamori. Dantas-Barbosaet a., FEMS Microbiol. Lett. 1998 169:185-190, describe transformationof Humicola grisea var. thernoidea to hygromycin B resistance, usingeither the lithium acetate method or electroporation.

[0016] Among the more successful fungal expression systems are those ofAspergillus and Trichoderma, for example as disclosed by Berka et al. inU.S. Pat. No. 5,578,463; see also Devchand and Gwynne, J. Biotechnol.17:3-9 (1991) and Gouka et al., Appl. Microbiol. Biotechnol. 47:1-11(1997). Examples of transformed strains of Myceliophthora thermophila,Acremonium alabamense, Thielavia terrestris and Sporotrichumcellulophilum are presented in WO 96/02563 and U.S. Pat. Nos. 5,602,004,5,604,129 and 5,695,985, which describe certain drawbacks of theAspergillus and Trichoderma systems and suggest that other fungi may bemore suited to large scale protein production.

[0017] Methods for the transformation of phyla other than Ascomycetesare known in the art; see for example Munoz-Rivas et al., Mol. Gen.Genet. 1986 205:103-106 (Schizophyllum commune); van de Rhee et al.,Mol. Gen. Genet. 1996 250:252-258 (Agaricus bisporus); Arnau et al.,Mol. Gen. Genet. 1991 225:193-198 (Mucor circinelloides); Liou et al.,Biosci. Biotechnol. Biochem. 1992 56:1503-1504 (Rhizopus niveus);Judelson et al., Mol. Plant Microbe Interact. 1991 4:602-607(Phytophthora infestans); and de Groot et al., Nature Biotechnol. 199816:839-842 (Agaricus bisporus).

[0018] In addition to the usual methods of transformation of filamentousfungi, such as for example protoplast fusion, Chakraborty and Kapoor,Nucleic Acids Res. 18:6737 (1990) describe the transformation offilamentous fungi by electroporation. De Groot et al., in NatureBiotechnol. 16:839-842 (1998), describe Agrobacteriumtumefaciens-mediated transformation of several filamentous fungi.Biolistic introduction of DNA into fungi has been carried out; see forexample Christiansen et al., Curr. Genet. 29:100-102 (1995); Durand etal., Curr. Genet. 31:158-161 (1997); and Barcellos et al., Can. J.Microbiol. 44:1137-1141 (1998). The use of magnetic particles for“magneto-biolistic” transfection of cells is described in U.S. Pat. Nos.5,516,670 and 5,753,477, and is expected to be applicable to filamentousfungi.

[0019] It is evident that much work has been done to develop expressionsystems using fungi as hosts. However, the common fin gal hosts are allfilamentous fungi, which tend to form entangled mats of mycelia inunstirred cultures, and highly viscous suspension (submerged) culturesin stirred tank bioreactors. These properties of filamentous fungi alsocause some problems in the industrial production of enzymes in fungalhost cells. For example, high viscosity and/or the local formation ofdense aggregates of mycelium, leads to difficulties in agitation,aeration, and nutrient diffusion. In general, filamentous fungi are notamenable to micropipetting of suspension cultures into microtiterplates, due to the viscosity of the cultures. Furthermore, due to theentangled mycelia, a culture of a typical filamentous fungus expressinga DNA library is not easily separated into separate clones on a largescale, which prevents evaluation of the individual genotypes as would berequired in a high-throughput assay system.

[0020] Typical filamentous fungi, in the absence of constant agitation,tend to grow in the form of mats on the surface of a liquid culturemedium, where they produce aerial spores. They do not generallysporulate when in submerged culture. Both of these properties presentsubstantial obstacles to the culture of filamentous fungal clones inmircotiter plates, and to the efficient manipulation and use of suchcultures for high-throughput screening. Suspended spores or otherreproductively competent elements would suitable for separation anddistribution into individual microtiter wells, whereas the production ofaerial spores will lead to cross-contamination of microtiter wells ifsurface mats are allowed to form. Agitation of the medium in microtiterwells, to the extent needed to prevent mat formation, is not feasible.In addition to the problem of difficult-to-control aerial spores,surface mats interfere with light transmission, making many assays (inparticular spectrophotometric absorbance assays) diffcult or impossible.Surface mats also interfere with processes such as oxygenation, reagentand nutrient addition, and pipetting.

[0021] The influence of fungal morphology on the physical properties ofthe culture has been recognized, and naturally-occurring strains havingmore favorable morphology have been identified, as described for exampleby Jensen and Boominathan in U.S. Pat. No. 5,695,985. Homogeneousdistribution of loose mycelium, with pronounced branching, was describedas a particularly desirable morphology. Schuster and Royer, ininternational patent application WO 97/26330 and U.S. Pat. No.6,184,026, suggest a similar method of identifying fungal cells havingmore suitable morphology for industrial production of heterologousproteins. The method comprises screening mutants of a parent fungal cellline, rather than wild-type strains, to find a specific alteredmorphology, transforming the mutant, and assessing whether a culture ofthe transformed mutant produces more heterologous protein than theparent cell line. Mutants with at least 10% greater hyphal branching areparticulary claimed. The method is illustrated for strains ofTrichoderma, Fusarium and Aspergillus, and is suggested to be applicableto numerous other genera.

[0022] The effect of branching frequency on culture viscosity ofAspergillus oryzae mutants was examined by Bocking et al., Biotechnol.Bioeng. 65:638-648 (1999); more highly branched strains exhibited lowerviscosity in this study. Van Wezel et al, in PCT application WO00/00613, describe methods for reducing the branching and/or enhancingthe fragmentation of filamentous microorganisms, whereby the viscosityof the culture is reduced. The method involves transforming themicroorganisms with the SsgA gene of Streptomyces griseus. The method isdemonstrated in filamentous bacteria of the order Actinomycetales, butis stated to be applicable to filamentous fungi. Dunn-Coleman et al., inWO 00/56893, describe an HbrA2 mutant A. nidulans, which exhibits ahyperbranched phenotype when grown above 42 ° C., and noted a linearrelationship between the degree of hyphal branching and cultureviscosity.

[0023] Most prior efforts in the field of filamentous fungal expressionsystems have been directed to the identification of strains suitable forindustrial production of enzymes, and therefore attention has beenfocused on culture viscosity, stability of transformation, yield ofheterologous protein per unit volume, and yield as a percentage ofbiomass. DNA libraries have been expressed in fungi; see for exampleGems and Clutterbuck, Curr. Genet. 1993 24:520-524, where an Aspergillusnidulans library was expressed in A nidulans and Gems et al., Mol. Gen.Genet. 1994 242:467-471 where a genomic library from Penicillium wasexpressed in Aspergillus. Neither of these reports disclosed orsuggested screening the expressed proteins; it was throughcomplementation of mutant alleles in the host that the expression ofgenes from the DNA library was demonstrated. The complementation methodrequires a specific mutant host for each exogenous protein activity onewishes to detect, and does not provide a tool for general libraryscreening.

[0024] The cloning of an Aspergillus niger invertase gene by expressionin Trichoderma reesei was described by Berges et al., Curr. Genet. 199324:53-59. Using an A. niger genomic library constructed in a cosmidvector containing a selectable marker, and using as the host T. reesei(which is incapable of utilizing sucrose), an A. niger invertase genewas cloned by a sib selection procedure. Here, again, a very specificcharacteristic of the host was required to detect the presence of asingle expressed exogenous protein, and screening of the genomic librarywas not disclosed or enabled.

[0025] The characteristics of a fugal host cell suitable for expressionof a DNA library are different in many respects from the characteristicsof hosts suitable for industrial protein manufacture. In general terms,a suitable fugal host for high-throughput screening should meet numerouscriteria; among them are the following:

[0026] The host must be transformed with high efficiency.

[0027] The host must process intron-containing genes and carry out anynecessary splicing.

[0028] The host must post-translationally process the expressed proteinso that it is produced in an active form.

[0029] Where the library is to be assayed for a protein, the host mustproduce the protein in high enough yield for detection by the assay.

[0030] The host should accept a variety of expression regulatoryelements, for ease of use and versatility.

[0031] The host should permit the use of easily-selectable markers.

[0032] The host cell cultures should be of low viscosity.

[0033] The host should be deficient in proteases and/or be anemable tosuppression of protease expression.

[0034] The host must permit screens for a wide variety of exogenousprotein activities or properties.

[0035] The hyphae in a culture of the host fungus should not be soentangled as to prevent the isolation of single clones, and should notbe so entangled as to raise the viscosity to the point of preventingefficient transfer and replication in a miniaturized high throughputscreening format (e.g. by micropipeting).

[0036] The host should not form surface mats, but should preferentiallygrow as a submerged culture.

[0037] The host should allow the efficient production of submergedspores or other propagules under the growth conditions provided in thehigh throughput screen.

[0038] In cases where metabolites are being screened for, it would beadvantageous if the host cells secreted the metabolites into the medium,where they could be readily detected and/or assayed. Ideally, the hostshould secrete only the exogenous protein.

[0039] In cases where a protein is being assayed for, it would beparticularly advantageous if the host also expressed enough heterologousprotein to enable isolation and purification of the protein. A host cellwith this characteristic would make it possible to further characterizeall heterologous proteins of interest merely by culturing the hostcells, without the time-consuming molecular biological manipulationsneeed to transfer the gene to another organism. Preferably, the hostshould be capable of secretion of the protein, as this would permit morereliable and more varied assays.

[0040] It would also be advantageous if the host cell were amenable toready isolation of the heterologous DNA, so that further studies andmodifications of the gene itself may be carried out.

[0041] In addition to these qualities of the host, the transformationsystem should also exhibit certrain characteristics. The transformationfrequency should be sufficiently high to generate the numbers oftransformants required for meaningful screens. Ideally, expression ofthe exogenous protein will be induced by a single inducer, by a singlepathway, acting on a single promoter.

[0042] To date, no combination of host cells and transformation systemhas been developed that meets all, or even most, of these criteria. Aneed therefore remains for fungal host cell and transformation systemsthat are capable of efficiently expressing the gene products of a DNAlibrary, especially genomic and/or eukaryotic genomic DNA libraries.

BRIEF DESCRIPTION OF THE INVENTION

[0043] The present invention employs filamentous fungi which produce“transferable reproductive elements” when grown in submerged culture. By“transferable reproductive element” is meant a spore, propagule, hyphalfragment, protoplast, micropellet, or other fungal element that is (1)readily separated from other such elements in the culture medium, and(2) capable of reproducing itself into a monoclonal culture. The fungipreferably also exhibit a less pronounced filamentous phenotype and/or acompact growth morphology, and produce low-viscosity cultures that aresuitable for the physical manipulations involved in high-throughput DNAlibrary screening. Particularly preferred are filamentous fungi which,even in the absence of agitation, tend to grow as submerged culturesrather than as surface mats.

[0044] The present invention takes advantage of the properties of thetransformation system disclosed in international patent applicationsPCT/NL99/00618 and PCT/EP99/202516. These applications describe anefficient transformation system for filamentous fungal hosts such asChrysosporium lucknowense and Aspergillus sojae. These applications alsodisclose that mutant strains are readily prepared which retain all theadvantages of the wild-type host cells, but which have partially losttheir filamentous phenotype and thus provide low-viscosity cultures.

[0045] The fungi preferred for use in the invention express and secretelarge amounts of exogenous protein, producing a high protein/biomassratio relative to previously known filamentous fungal hosts. Theinvention provides a transformation system that exhibits high yields oftransformants. The invention also provides libraries of transformantfungi which efficiently express the protein products of heterologouscDNA inserts, and especially genomic DNA inserts. In another aspect ofthe invention, the libraries of transformed fungi may be used inscreening for activities or properties of the heterologous proteins, orin screening for metabolites produced by the transformed fungi as aconsequence of exogenous protein activities, or in screening for theheterologous DNA or for RNA transcripts derived therefrom. It will beappreciated that the present invention also enables high-throughputscreening for metabolites of non-transformed strains having thephenotypic characteristics described above.

[0046] The term “mutant filamentous fungus” as used herein refers simplyto fungi not found in nature. The “mutations” that lead to desirablephenotypic characteristics, such as a compact growth form, lowviscosity, reduced protease levels, submerged growth, etc., may beintroduced randomly by either classical means, such as UV irradiationand chemical mutagenesis, or by molecular biological methods such ascassette mutagenesis, or may be deliberately introduced by geneticengineering methods. Should a naturally-occurring fungus be found topossess the necessary properties, it will of course be usable in themethods of the invention.

[0047] In yet another aspect of the invention, the libraries oftransformed fungi may be screened for useful properties of the fungithemselves, such as for example high levels of production of aparticular expressed protein or metabolite. This aspect of the inventionis illustrated by a quantitative assay for the expressed protein ofinterest, where the particular transformant having the most favorablecombination of protein production, protein processing, and proteinsecretion would be detected.

[0048] In another aspect of the invention, the libraries of transformedfungi may be screened for the presence of DNA sequences capable ofhybridizing to a nucleic acid probe of interest.

DESCRIPTION OF THE FIGURES

[0049]FIG. 1 is a Western blot as described in the Examples.

[0050]FIG. 2 is a pUT720 map.

[0051]FIG. 3 is a pUT970G map.

[0052]FIG. 4 is a pUT1064 map.

[0053]FIG. 5 is a pUT1065 map.

[0054]FIG. 6 is a pF6g map.

[0055]FIG. 7 is a pUT1150 map.

[0056]FIG. 8 is a pUT1152 map.

[0057]FIG. 9 is a pUT1155 map.

[0058]FIG. 10 is a pUT1160 map.

[0059]FIG. 11 is a pUT 1162 map.

[0060]FIG. 12 is the schematic structure of the pclA protein.

[0061]FIG. 13A is a photomicrograph of wildtype Aspergillus niger.

[0062]FIG. 13B is a photomicrobraph of an Aspergillus niger pclA mutant.

[0063]FIG. 14A is a photomicrograph of wildtype Aspergillus sojae.

[0064]FIG. 14B is a photomicrobraph of an Aspergillus sojae pclA mutant.

[0065] FIGS. 15A-E present sequencing results of the pyrE gene.Underlining indicates amino acid sequence; it is not continuous due tosome sequence uncertainties. The indicated amino acids are the mostprobable. Bold type indicates putative/probable introns.

DETAILED DESCRIPTION OF THE INVENTION

[0066] In its broadest aspect, the invention is directed to transformedfilamentous fungi that generate transferable reproductive elements insuspension, to libraries of such fungi, and to methods of screening suchlibraries for biological properties of interest, such as biochemical orbiological activity associated with expressed exogenous proteins orassociated with metabolites, i.e. small molecule products produced byendgoenous and/or exogenous enzymes. The library of low-viscosityfilamentous fungi comprises fungi containing nucleic acid sequences,each nucleic acid sequence encoding a heterologous protein, each of saidnucleic acid sequences being operably linked to an expression regulatingregion and optionally a secretion signal encoding sequence and/or acarrier protein encoding sequence. Preferably a transformed strainaccording to the invention will secrete the heterologous protein.

[0067] The expression and screeing methods of the invention, and thefungi employed therein, are useful for producing fungi, proteins,metabolites, and DNA molecules having utility in a variety ofapplications. The methods of the invention are also useful for producingnucleic acid and protein sequence information, and this informationitself is regarded as a valuable product of the claimed methods.

[0068] Preferred filamentous fungi of the invention are characterized bythe low viscosity of the culture medium. Whereas a typicalindustrial-grade filamentous fungus will produce cultures withviscosities well over 200 centipoise (cP) and usually over 1,000 cP, andcan reach 10,000 cP, the fungi of this invention exhibit a cultureviscosity of less than 200 cP, preferably less than 100 cP, morepreferably less than 60 cP, and most preferably less than 10 cP after 48or more hours of culturing in the presence of adequate nutrients underoptimal or near-optimal growth conditions. The filamentous fungi of theinvention usually exhibit a morphology characterized by short, discrete,non-entangled hyphae, or micropellets. Micropellets are slightly- ornon-entangled collections of hyphae arising from a single clone, asdistinct from pellets which are much larger and are derived frommultiple entangled clones. For example, the mutant UV18-25 Chrysosporiumlucknowense strain (viscosity<10 cP ) and the morphologically similarmutant Trichoderma longibrachiatum X-252 strain (viscosity<60 cP ) arecharacterised by the presence of short, distinct, non-entangled hyphaebetween 100 and 200 microns in length, and the low viscosity engineeredmutant Aspergillus sojae pclA is characterized by a compact form withconsiderable branching and short hyphae (see FIG. 14). Whereas thelow-viscosity fungi described in WO97/26330 are described as having“more extensive hyphal branching,” some fungi of the present inventionhave equivalent or even slightly reduced hyphal branching when comparedto the non-mutant strains. It appears that hyphal length plays thedominant role in controlling the viscosity of the culture.

[0069] Particularly preferred fungal strains are characterized by havinga high exogenous secreted protein/biomass ratio. This ratio ispreferably greater than 1:1, more preferably greater than 2:1, and evenmore preferably 6:1 or greater. Most preferably, the ratio is 8:1 orhigher. Such high ratios are advantageous in a high-throughput screeningenvironment, because they result in a higher concentration of exogenousprotein, allowing more sensitive and/or more rapid screening assays.This is of particular benefit as the volume of the assay solutiondecreases, for example upon going from 96-well plates to 384-wellplates, and thence to 1536-well plates. The methods of the presentinvention are suitable for any of these microtiter plate formats, andfor most other HTS formats employing liquid samples.

[0070] It is contemplated that any filamentous fungus can be converted,by the processes of mutation described herein, into mutant strainssuitable for use in the present invention. Among the preferred genera offilamentous fungi are the Chrysosporium, Thielavia, Neurospora,Aureobasidium, Filibasidium, Piromyces, Cryplococcus, Acremonium,Tolypocladium, Scytalidium, Schizophyllum, Sporotrichum, Penicillium,Gibberella, Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, andTrichoderma, and anamorphs and teleomorphs thereof. More preferred areChrysosporium, Trichoderma, Aspergillus, and Fusarium. Most preferred isChrysosporium. The genus and species of fungi can be defined bymorphology consistent with that disclosed in Barnett and Hunter,Illustrated Genera of Imperfect Fungi, 3rd Edition, 1972, BurgessPublishing Company. A source providing details concerning classificationof fungi of the genus Chrysosporium is Van Oorschot, C. A. N. (1980) “Arevision of Chrysosporium and allied genera” in Studies in Mycology No.20, Centraal Bureau voor Schimmelcultures (CBS), Baarn, The Netherlands,pp. 1-36. According to these teachings the genus Chrysosporium fallswithin the family Moniliaceae which belongs to the order Hyphomycetales.

[0071] Another ready source providing information on fungal nomenclatureare the Budapest Treaty depositories, especially those providing onlinedatabases (the following internet addresses employ the http protocol).The ATCC (US) provides information at www.atcc.org, the CBS (NE) atwww.cbs.knaw.nl, and the VKM (RU) atwww.bdt.org.br.bdt.msdn.vkm/general. Another source isNT.ars-grin.gov/fungaldatabases. All these institutions can provideteaching on the distinguishing characteristics of fungal species. Analternate taxonomy of the Ascomycota may be found atwww.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Undef&id=4890.According to this alternate taxonomy, the genus Chrysosporium belongs tofamily Onygenaceae, order Onygenales, phylum Ascomycota.

[0072] The definition of Chrysosporium includes but is not limited tothese strains: C. botryoides, C. carmichaeli, C. crassitunicatum, C.europae, C. evolceannui, C. farinicola, C. fastidium, C. filiforme, C.georgiae, C. globiferum, C. globiferum var. articulatum, C. globiferumvar. niveum, C. hirundo, C. hispanicum, C. holmii, C. indicum, C. iops,C. keratinophilum, C. kreiselii, C. kuzurovianum, C. lignorum, C.obatum, C. lucknowense, C. lucknowense Garg 27K, C. medium, C. mediumvar. spissescens, C. mephiticum, C. merdarium, C. merdarium var. roseum,C. minor, C. pannicola, C. parvum, C. parvum var. crescens, C. pilosum,C. pseudomerdarium, C. pyriformis, C. queenslandicum, C. sigleri, C.sulfureum, C. synchronum, C. tropicum, C. undulatum, C. vallenarense, C.vespertilium, C. zonatum.

[0073]C. lucknowense is a species of Chrysosporium that is of particularinterest as it has provided a natural high producer of cellulaseproteins (international applications WO 98/15633, PCT/NL99/00618, andU.S. Pat. Nos. 5,811,381 and 6,015,707). Strains with internationaldepository accession numbers ATCC 44006, CBS 251.72, CBS 143.77, CBS272.77, and VKM F-3500D are examples of Chrysosporium lucknowensestrains. Also included within the definition of Chrysosporium arestrains derived from Chrysosporium predecessors including those thathave mutated either naturally or by induced mutagenesis. The methods ofthe invention, in one embodiment, employ mutants of Chrysosporium,obtained by a combination of irradiation and chemically-inducedmutagenesis, that tend to produce transferable reproductive elements insuspension, and that exhibit a morphology characterized by short,discrete, non entangled hyphae (“compact growth”), and a phenotypecharacterized by submerged growth and reduced viscosity of thefermentation medium when cultured in suspension. In another embodiment,the invention employs phenotypically similar mutants of Trichoderma. Inyet other embodiments the invention employs phenotypically similarmutants of Aspergillus sojae or Aspergillus niger.

[0074] For example, VKM F-3500D (strain “C1”) was mutagenised bysubjecting it to ultraviolet light to generate strain UV13-6. Thisstrain was subsequently further mutated withN-methyl-N′-nitro-N-nitrosoguanidine to generate strain NG7C-19. Thelatter strain in turn was subjected to mutation by ultraviolet lightresulting in strain UV18-25 (VKM F-3631 D). During this mutation processthe morphological characteristics varied somewhat in culture in liquidor on plates as well as under the microscope. With each successivemutagenesis the cultures showed less of the fluffy and felty appearanceon plates that are described as being characteristic of Chrysosporium,until the colonies attained a flat and matted appearance. A brownpigment observed with the wild type strain in some media was lessprevalent in mutant strains. In liquid culture the mutant UV18-25 wasnoticeably less viscous than the wild type strain C1 and the mutantsUV13-6 and NG7C-19. While all strains maintained the gross microscopiccharacteristics of Chrysosporium, the mycelia became narrower with eachsuccessive mutation and with UV18-25 distinct fragmentation of themycelia could be observed. This mycelial fragmentation is likely to be acause of the lower viscosity associated with cultures of UV1 8-25. Thecapacity of the strains for aerial sporulation decreased with eachmutagenic step. These results demonstrate that a strain may belonggenetically to the genus Chrysosporium while exhibiting deviations fromthe traditional taxonomic (morphological) definitions.

[0075] In particular the anamorph form of Chrysosporium has been foundto be suited for the screening application according to the invention.The metabolism of the anamorph renders it particularly suitable for ahigh degree of expression. A teleomorph should also be suitable as thegenetic make-up of the anamorphs and teleomorphs is identical. Thedifference between anamorph and teleomorph is that one is the asexualstate and the other is the sexual state; the two states exhibitdifferent morphology under certain conditions.

[0076] Another example embodies genetically engineered mutant strains ofAspergillus sojae. In one of these mutants a specific endoproteaseencoding gene was disrupted. This resulted in a compact growth phenotypeexhibiting enhanced branching and short hyphae, and the formation ofmicropellets in submerged cultivation. Moreover, the Aspergillus sojaereferred to in this application may be induced to exhibit efficientsporulation under specific submerged cultivation conditions, whichrenders it especially suitable for use in a high-throughput screeningsystem. In this case, the conditions conducive to formation of thetransferable reproductive elements simply consisted of a syntheticmedium containing 0.6 g/ml EDTA. The conducive conditions will vary fromone host to another, but it is evident that the conditions will alreadybe known if a host has been found to be suitable.

[0077] It is preferable to use non-toxigenic and non-pathogenic fungalstrains, of which a number are known in the art, as this will reducerisks to the practitioner and will simplify the overall screeningprocess. In a preferred embodiment the fungi will also be proteasedeficient, so as to minimize degradation of the exogenous proteins,and/or amenable to suppression of protease production. The use ofprotease deficient strains as expression hosts is well known; see forexample PCT application WO 96/29391. Protease deficient strains may beproduced by screening of mutants, or the protease gene(s) may be“knocked out” or otherwise inactivated by methods known in the art, asdescribed for example by Christensen and Hynes in U.S. Pat. No.6,025,185 (Aspergillus oryzae with non-functional areA gene).

[0078] It has been found that Chrysosporium mutants can be made thathave reduced expression of protease, thus making them even more suitablefor the production of proteinaceous products, especially if theproteinaceous product is sensitive to protease activity. Thus theinvention my also employ a mutant Chrysosporium strain which producesless protease than non-mutant Chrysosporium strain, for example lessthan C. lucknowense strain C1 (VKM F-3500 D). In particular the proteaseacitivity (other than any selective protease intended to cleave asecreted fusion protein) of such strains is less than half the amount,more preferably less than 30% of the amount, and most preferably lessthan about 10% the amount produced by the C1 strain. The decreasedprotease activity can be measured by known methods, such as by measuringthe halo formed on skim milk plates or by bovine serum albumin (BSA)degradation.

[0079] It may be desirable to inactivate other genes in the hostfilamentous fungus, such as for example those encoding cellulases andother heavily secreted proteins, in order to minimize interference inthe assay by host proteins. The genes encoding secreted proteins may bedeleted or mutated, or alternatively genes controlling the inductionsystem or other pathways involved in the expession of unwanted proteinsmay be modified in such a way as to reduce such expression. Where anendogenous promoter is employed in the vectors of the invention (seebelow), it may be especially desirable to inactivate genes for otherproteins under control of the same inducer. Fungi amenable tosuppression of protease secretion are those where protease expression isunder the control of a regulatory element that responds to environmentalconditions, such that these conditions (e.g., amino acid concentration)can be manipulated to minimize protease production.

[0080] Preferably a homologous expression-regulating region enablinghigh expression in the selected host is employed in the transformingvector. High expression-regulating regions derived from a heterologoushost, such as from Trichoderma or Aspergillus, are well known in the artand can also be used. By way of example, and not limitation, examples ofproteins known to be expressed in large quantities and thus providingsuitable expression regulating sequences for use in the presentinvention are hydrophobin, protease, amylase, xylanase, pectinase,esterase, beta-galactosidase, cellulase (e.g. endo-glucanase,cellobiohydrolase) and polygalacturonase.

[0081] An expression-regulating region comprises a promoter sequenceoperably linked to a nucleic acid sequence encoding the protein to beexpressed. The promoter is linked such that the positioning vis-à-visthe initiation codon of the sequence to be expressed allows expression.The promoter sequence can be constitutive but preferably is inducible.Use of an inducible promoter and appropriate induction media favorsexpression of genes operably linked to the promoter. Any expressionregulating sequence from a homologous species, or from a heterologousstrain capable of permitting expression of a protein, is envisaged. Theexpression regulating sequence is suitably a fungalexpression-regulating region, e.g. an ascomycete regulating region.Suitably the ascomycete expression regulating region is a regulatingregion from any of the following genera: Aspergillus, Trichoderma,Chrysosporium, Humicola, Neurospora, Tolypocladium, Fusarium,Penicillium, Talaromyces, or alternative sexual forms thereof such asEmericela and Hypocrea. The cellobiohydrolase promoter from Trichoderma;alcohol dehydrogenase A, alcohol debydrogenase R, glutamatedehydrogenase, TAKA amylase, glucoamylase, and glyceraldehyde phosphatedehydrogenase promoters from Aspergillus; phosphoglycerate andcross-pathway control promoters of Neurospora; lipase and asparticproteinase promoter of Rhizomucor miehei; beta-galactosidase promoter ofPenicillium canescens; and cellobiohydrolase, endoglucanase, xylanase,glyceraldehyde-3-phosphate dehydrogenase A, and protease promoters fromChrysosporium are representative examples. An expression regulatingsequence from the same genus as the host strain is preferable, as it ismore likely to be specifically adapted to the host.

[0082] Natural expression-regulating sequences from strains ofChrysosporium which express proteins in extremely large amounts, areparticularly preferred. Examples of such strains have been deposited inaccordance with the Budapest Treaty with the All Russian Collection(VKM) depository institute in Moscow. Wild type C1 strain has the numberVKM F-3500 D, deposit date Aug. 29, 1996, C1 UV13-6 mutant was depositedwith number VKM F-3632 D, and deposit date Sep. 02, 1998, C1 NG7C-19mutant was deposited with number VKM F-3633 D and deposit date Sep. 02,1998 and C1 UV18-25 mutant was deposited with number VKM F-3631 D anddeposit date Sep. 02, 1998. These strains are also preferred as sourcesfor the generation of low-viscosity mutants; indeed the VKM F-3631 Dstrain already exhibits the necessary low viscosity phenotype. Alow-viscosity mutant Trichoderma strain, designated X-252, was obtainedafter two rounds of irradiation of Trichoderma longibrachiatum 18.2KK,which in turn was derived by mutation of the QM 9414 strain of T.longibrachiatum (ATCC 26921). In other embodiments the invention employsphenotypically similar mutants of Aspergillus sojae and Aspergillusniger.

[0083] Preferably, where the host is a Chrysosporium, a Chrysosporiumpromoter sequence is employed to ensure good recognition thereof by thehost. Certain heterologous expression-regulating sequences also work asefficiently in Chrysosporium as native Chrysosporium sequences. Thisallows well-known constructs and vectors to be used in transformation ofChrysosporium, and offers numerous other possibilities for constructingvectors enabling good rates of transformation and expression in thishost. For example, standard Aspergillus transformation techniques can beused as described for example by Christiansen et al. in Bio/Technology1988 6:1419-1422. Other documents providing details of Aspergillustransformation vectors, e.g. U.S. Pat. Nos. 4,816,405, 5,198,345,5,503,991, 5,364,770, 5,705,358, 5,728,547, and 5,578,463, EP-B-215.594(also for Trichoderma) and their contents are incorporated by reference.As extremely high expression rates for cellulase have been observed inChrysosporium strains, the expression regulating regions of cellulasegenes are particularly preferred.

[0084] The vectors of the invention can comprise a promoter sequencederived from a gene encoding an enzyme, preferably a secreted enzyme.Examples of suitable enzymes from which promoter sequences may be takenare the carbohydrate-degrading enzymes (e.g., cellulases, xylanases,mannanases, mannosidases, pectinases, amylases, e.g. glucoamylases,α-amylases, α- and βp-galactosidases, α- and β-glucosidases,β-glucanases, chitinases, chitanases), proteases (endoproteases,amino-proteases, amino-and carboxy-peptidases), other hydrolases(lipases, esterases, phytases), oxidoreductases (catalases,glucose-oxidases) and transferases (transglycosylases,transglutaminases, isomerases and invertases). Several examples fromChrysosporium lucknowense are presented in Table A.

[0085] A nucleic acid construct will preferably comprise a nucleic acidexpression regulatory region from Chrysosporium, more preferably fromChrysosporium lucknowense or a derivative thereof, operably linked to anucleic acid sequence encoding a protein to be expressed. Particularlypreferred nucleic acid constructs will comprise an expression regulatoryregion from Chrysosporium associated with cellulase or xylanaseexpression, preferably cellobiohydrolase expression, most preferablyexpression of the 55 kDa cellobiohydrolase (CBH1) described in Table A.As additional examples, the Chrysosporium promoter sequences ofhydrophobin, protease, amylase, xylanase, esterase, pectinase,beta-galactosidase, cellulase (e.g. endoglucanase, cellobiohydrolase)and polygalacturonase are also considered to fall within the scope ofthe invention. TABLE A Characteristics of selected enzymes fromChrysosporium lucknowense Stability Highest pH at which >50% Highest pHat which >70% 20 h, 50° C. activity is retained activity is retained pH7.5/8 No. of RBB Other RBB Other % of max amino CMC CMC sub- CMC CMCsub- activity Sample acids ase ase strates ase ase strates remaining 30Kd alkaline protease — — 12.5  — — 12.0  — 30 kD Xyl (alkaline) 333 — —10.0  — — 8.5 80 51 kD Xyl — — 8.0 — — 7.5 — 60 kD Xyl — — 9.5 — — 9.085 30 kD endo (EG3) 247 45 kD endo 7.0 8.0 — 6.5 7.0 — 75 55 kD endo 2478.0 8.0 — 7.0 7.0 — 55 25 kD(21.8 kD)endo (EG5) 225 7.5 10.0  — 6.5 9.0— 80 43 kD(39.6 kD*)endo (EG6) 395 8.0 8.0 — 7.2 7.2 — — 45 kDα,β-Gal/β-Gluc — — 6.8 — — 5.7 — 48 kD CBH 5.2 7.5 8.0 5.0 6.8 — — 55 kDCBH1 526 8.0 9.0 — 7.4 8.5 — 70 65 kD PGU — — 8.0 — — 7.3 — 90 kDprotease — — 9.0 — — 9.0 — 100 kD esterase — — 9.0 — — 9.0 —

[0086] Any of the promoters or regulatory regions of expression ofenzymes disclosed in Table A, for example, can be suitably employed. Thenucleic acid sequences of these promoters and regulatory regions canreadily be obtained from a Chrysosporium strain. Methods by whichpromoter sequences can be determined are numerous and well known in theart. Promoter sequences are generally found immediately preceding theATG start codon at the beginning of the relevant gene. For example,promoter sequences can be identified by deleting sequences upstream ofthe relevant gene, using recombinant DNA techniques, and examining theeffects of these deletions on expression of the gene. Also, for example,promoter sequences can often be inferred by comparing the sequence ofregions upstream of the relevant gene with concensus promoter sequences.

[0087] For example, the promoter sequences of C1 endoglucanases wereidentified in this manner (see PCT/NL99/00618) by cloning thecorresponding genes. Preferred promoters according to the invention arethe 55 kDa cellobiohydrolase (CBH1), glyceraldehyde-3-phosphatedehydrogenase A, and the 30 kDa xylanase (Xy1F) promoters fromChrysosporium, as these enzymes are expressed at high level by their ownpromoters. The promoters of the carbohydrate-degrading enzymes ofChrysosporium lucknowense in particular, especially C. lucknowense GARG27K, can advantageously be used for expressing libraries of proteins inother fungal host organisms.

[0088] Particular embodiments of nucleic acid sequences according to theinvention are known for Chrysosporium, Aspergillus and Trichoderma.Promoters for Chrysosporium are described in PCT/NL99/00618. The priorart provides a number of expression regulating regions for use inAspergillus, e.g. U.S. Pat. Nos. 4,935,349; 5,198,345; 5,252,726;5,705,358; and 5,965,384; and PCT application WO 93/07277. Expression inTrichoderma is disclosed in U.S. Pat. No. 6,022,725. The contents ofthese patents are hereby incorporated by reference in their entirety.

[0089] The hydrophobin gene is a fungal gene that is highly expressed.It is thus suggested that the promoter sequence of a hydrophobin gene,preferably from Chrysosporium, may be suitably applied as expressionregulating sequence in a suitable embodiment of the invention.Trichoderma reesei and Trichoderma harzianum gene sequences forhydrophobin have been disclosed for example in the prior art as well asa gene sequence for Aspergillus fumigatus and Aspergillus nidulans andthe relevant sequence information is hereby incorporated by reference(Nakari-Setala et al., Eur. J. Biochem. 1996, 235:248-255; Parta et al.,Infect. Immun. 1994 62:4389-4395; Munoz et al., Curr. Genet. 1997,32:225-230; and Stringer et al., Mol. Microbiol. 1995 16:33-44). Usingthis sequence information a person skilled in the art can obtain theexpression regulating sequences of Chrysosporium hydrophobin geneswithout undue experimentation following standard techniques such asthose suggested above. A recombinant Chrysosporium strain according tothe invention can comprise a hydrophobin-regulating region operablylinked to the sequence encoding the heterologous protein.

[0090] An expression regulating sequence can also additionally comprisean enhancer or silencer. These are also well known in the prior art andare usually located some distance away from the promoter. The expressionregulating sequences can also comprise promoters with activator bindingsites and repressor binding sites. In some cases such sites may also bemodified to eliminate this type of regulation. For example, filamentousfungal promoters in which creA sites are present have been described.The creA sites can be mutated to ensure that the glucose repressionnormally resulting from the presence of creA is eliminated. Use of sucha promoter enables production of the library of proteins encoded by thenucleic acid sequences regulated by the promoter in the presence ofglucose. The method is exemplified in WO 94/13820 and WO 97/09438. Thesepromoters can be used either with or without their creA sites. Mutantsin which the creA sites have been mutated can be used as expressionregulating sequences in a recombinant strain according to the inventionand the library of nucleic acid sequences it regulates can then beexpressed in the presence of glucose. Such Chrysosporium promotersensure derepression in an analogous manner to that illustrated in WO97/09438. The identity of creA sites is known from the prior art.Alternatively, it is possible to apply a promoter with CreA bindingsites that have not been mutated in a host strain with a mutationelsewhere in the repression system e.g. in the creA gene itself, so thatthe strain can, notwithstanding the presence of creA binding sites,produce the library of proteins in the presence of glucose.

[0091] Terminator sequences are also expression-regulating sequences andthese are operably linked to the 3′ termini of the sequences to beexpressed. A variety of known fungal terminators are likely to befunctional in the host strains of the invention. Examples are the A.nidulans trpC terminator, A. niger alpha-glucosidase terminator, A.niger glucoamylase terminator, Mucor miehei carboxyl protease terminator(see U.S. Pat. No. 5,578,463), and the Trichoderma reeseicellobiohydrolase terminator. Chrysosporium terminator sequences, e.g.the EG6 terminator, will of course function well in Chrysosporium.

[0092] A suitable transformation vector for use according to theinvention may optionally have the exogenous nucleic acid sequences to beexpressed operably linked to a sequence encoding a signal sequence. Asignal sequence is an amino acid sequence which, when operably linked tothe amino acid sequence of an expressed protein, enables secretion ofthe protein from the host organism. Such a signal sequence may be oneassociated with a heterologous protein or it may be one native to thehost. The nucleic acid sequence encoding the signal sequence must bepositioned in frame to permit translation of the signal sequence and theheterologous proteins. Signal sequences will be particularly preferredwhere the invention is being used in conjunction with directed molecularevolution, and a single, secreted exogenous protein is being evolved.

[0093] It will be understood that it is less advanatageous toincorporate a signal sequence in a vector that is to be used to expressa library, as this will decrease the probability of expressing theprotein of interest. In a genomic library prepared by randomly shearingthe DNA and cloning into a vector, the probability that one would obtainan in frame fusion of a gene in the library to the signal sequence islow. Also, even where an in-frame fusion has been obtained, the chosensignal sequence may not work with all genes. For these reasons it may bepreferable not to employ a signal sequence when screening a genomic DNAlibrary, but rather to screen for the activity or presence ofintracelllular exogenous protein. Analysis of the activity or presenceof intracellular proteins may be accomplished by pretreating thetransformant library with enzymes that convert the fungal cells toprotoplasts, followed by lysis. The procedure has been described by vanZeyl et al., J. Biotechnol. 59:221-224 (1997). This procedure has beenapplied to Chrysosporium to allow colony PCR from Chrysosporiumtransformants grown in microtiter plates.

[0094] Any signal sequence capable of permitting secretion of a proteinfrom a Chrysosporium strain is envisaged. Such a signal sequence ispreferably a fungal signal sequence, more preferably an Ascomycetesignal sequence. Suitable signal sequences can be derived fromeukaryotes generally, preferably from yeasts or from any of thefollowing genera of fungi: Aspergillus, Trichoderma, Chrysosporium,Pichia, Neurospora, Rhizomucor, Hansenula, Humicola, Mucor,Tolypocladium, Fusarium, Penicillium, Saccharomyces, Talaromyces oralternative sexual forms thereof such as Emericella and Hypocrea. Signalsequences that are particularly useful are those natively associatedwith cellobiohydrolase, endoglucanase, beta-galactosidase, xylanase,pectinase, esterase, hydrophobin, protease or amylase. Examples includeamylase or glucoamylase of Aspergillus or Humicola, TAKA amylase ofAspergillus oryzae, α-amylase of Aspergillus niger, carboxyl peptidaseof Mucor (U.S. Pat. No. 5,578,463), a lipase or proteinase fromRhizomucor miehei, cellobiohydrolase of Trichoderma, beta-galactosidaseof Penicillium canescens CBH1 from Chrysosporium, and the alpha matingfactor of Saccharomyces.

[0095] Alternatively the signal sequence can be from an amylase orsubtilisin gene of a strain of Bacillus. A signal sequence from the samegenus as the host strain is extremely suitable as it is most likely tobe specifically adapted to the specific host; thus when Chrysosporiumlucknowense is the host, the signal sequence is preferably a signalsequence of Chrysosporium. Chrysosporium strains C1, UV13-6, NG7C-19 andUV18-25 secrete proteins in extremely large amounts, and signalsequences from these strains are of particular interest. Signalsequences from filamentous fungi and yeast may be useful, as well assignal sequences of non-fungal origin.

[0096] A transformed recombinant host fungus according to any of theembodiments of the invention can further comprise a selectable marker.Such a selectable marker permits selection of transformed or transfectedcells. A selectable marker often encodes a gene product providing aspecific type of resistance foreign to the non-transformed strain. Thiscan be resistance to heavy metals, antibiotics or biocides in general.Prototrophy is also a useful selectable marker of the non-antibioticvariety. Auxotrophic markers generate nutritional deficiencies in thehost cells, and genes correcting those deficiencies can be used forselection. Examples of commonly used resistance and auxotrophicselection markers are amdS (acetamidase), hph (hygromycinphosphotransferase), pyrG (orotidine-5′-phosphate decarboxylase), andpyrE (orotate P-ribosyl transferase, trpC (anthranilate synthase), argB(ornithine carbamoyltransferase), sC (sulphate adenyltransferase), bar(phosphinothricin acetyltransferase), niaD (nitrate reductase), Sh-ble(bleomycin-phleomycin resistance), mutant acetolactate synthase(sulfonylurea resistance), and neomycin phosphotransferase(aminoglycoside resistance). A preferred selection marker inChrysosporium is orotate P-ribosyl transferase. Selection can be carriedout by cotransformation where the selection marker is on a separatevector or where the selection marker is on the same nucleic acidfragment as the protein-encoding sequence for the heterologous protein.

[0097] A further improvement of the transformation frequency may beobtained by the use of the AMA1 replicator sequence, which is useful forexample in Aspergillus niger (Verdoes et al., Gene 146:159-165 (1994)).This sequence results in a 10- to 100-fold increase in thetransformation frequency in a number of different filamentous fungi.Furthermore, the introduced DNA is retained autonomously in the fungalcells, in a multiple-copy fashion, without integration into the fungalgenome. This is expected to be beneficial for the high throughputscreening method of the present invention, as the non-integrative statereduces variations in the level of gene expression between differenttransformants. Moreover, as the introduced DNA is not recombined intothe host DNA, no unwanted mutations in the host genome will occur.Uniform levels of exogenous gene expression may be obtained by use ofautonomously replicating vectors such as AMA1, or alternatively,autonomous replication in fungi can be promoted by telomeric sequences(see e.g. A. Aleksenko and L. Ivanova, Mol. Gen. Genet. 1998260:159-164.)

[0098] As used herein the term “heterologous protein” is a protein orpolypeptide not normally expressed or secreted by the host strain usedfor expression according to the invention. A heterologous protein may beof prokayotic origin, or it may be derived from a fungus, plant, insect,or higher animal such as a mammal. For pharmaceutical screening purposesquite often a preference will exist for human proteins, thus a preferredembodiment will be a host wherein the DNA library is of human origin.Such embodiments are therefore also considered suitable examples of theinvention.

[0099] Expression of a library of human genes, derived from a genomichuman DNA library, in the filamentous fungi of the invention is expectedto be efficient for several reasons. It is now known that the averagesize of human genes is 3,000-5,000 bp, and that human introns averageabout 75 to about 150 bp (total range 40- >50,000). Filamentous fungihave introns of 40-75 bp, but they can deal with introns up to 500 bp inlength. On average, human genes carry 3-5 introns per gene (M. Deutsch,M. Long, Nucl. Acids Res. 1999 27:3219-3228; Table B). Human signalsequences are also known to function in filamentous fungi. For thesereasons, it is likely that a large number of human genes can beexpressed and secreted at high levels by the methods of this invention.TABLE B In- trons Organ- per Average intron ism gene size (nt) (range)Intron structure Animal/ 3-5  75-150 GTnnGt . . . CtxAC . . . yAG Plant    (40->50000) 80% under 150 nt Fungi 3 40-75 GTAnGy . . . CtxAC . . .yAG  (40-500) Yeast 0.01 50-60 GTATGT . . . TACTAAC . . . yAG (?—?)

[0100] The methods of the invention are thus expected to be useful forexpression of DNA libraries derived from both prokaryotic and eukaryoticgenomes. As described above, the methods are capable of expression anddiscovery of both secreted and intracellular proteins, giving readyaccess to an extemely large number of genes and proteins.

[0101] A further aspect of the invention includes the construction andscreening of fungal mutant libraries, and fungal mutant librariesprepared by the methods disclosed herein. The libraries may be obtainedby transformation of the fungal hosts according to this invention withany means of integrative or non-integrative transformation, usingmethods known to those skilled in the art. This library of fungi basedon the preferred host strains may be handled and screened for desiredproperties or activities of exogenous proteins in miniaturized and/orhigh-throughput format screening methods. By property or activity ofinterest is meant any physical, physicochemical, chemical, biological,or catalytic property, or any improvement, increase, or decrease in sucha property, associated with an exogenous protein of a library member.The library may also be screened for metabolites, or for a property oractivity associated with a metabolite, produced as a result of thepresence of exogenous and/or endogenous proteins. The library may alsobe screened for fungi producing increased or decreased quantities ofsuch protein or metabolites.

[0102] In another aspect of this invention, the library of transformedfungi may be screened for the presence of fungal metabolites havingdesirable properties. Examples of such metabolites include polyketides,alkaloids, and terpenoid natural products. It is anticipated thatmultiple genes or gene clusters (operons) may be transferred to the hostcells of the invention, and that non-protein products generated by theaction of the encoded enzymes will then be generated in the host cells.For example, it has been shown that DNA encoding the proteins necessaryfor production of lovastatin can be transferred to Aspergillus oryzae(U.S. Pat. No. 5,362,638; see also U.S. Pat. No. 5,849,541).

[0103] In another emodiment of the invention, the library of transformedfungi may be screened for the presence of DNA that hybridizes to anucleic acid probe of interest. In this embodiment, expression and/orsecretion of exogenous proteins is not essential, although it will oftenstill be desirable. Where protein expressin is not needed, it will beappreciated that regulatory sequences are not needed in the vector.

[0104] In yet another embodiment of the invention, the library oftransformed fungi may be screened for some desirable property of thefungi themselves, such as for example tolerance to a physically orchemically extreme environment, or the ability to produce, modify,degrade or metabolize a substance of interest. Such desirable propertiesmay or may not be ascribable to the presence of a single exogenousprotein. This embodiment will be of particular utility when employed aspart of a process of directed evolution.

[0105] The heterologous DNA may be genomic DNA or cDNA, prepared frombiological specimens by methods well known in the art. The biologicalspecimen may be an environmental sample (for example, soil, compost,forest litter, seawater, or fresh water), or an extracted, filtered, orcentrifuged or otherwise concentrated sample therefrom. Mixed culturesof microorganisms derived from environmental samples may be employed aswell. The biological sample may also be derived from any single speciesof organism, such as a cultured microorganism, or plant, insect, orother animal such as a mammal. In addition, the heterologous DNA may besynthetic or semi-synthetic, for example random DNA sequences or DNAcomprising naturally-occurring segments which have been shuffled,mutated, or otherwise altered. An example of a semi-synthetic nucleiclibrary is found in Wagner et al., WO 00/0632. DNA from environmentalsamples (or mixed cultures derived therefrom) will be advantageous forthe discovery of novel proteins, while the use of DNA from a singlespecies will be advantageous in that (1) an appropriate vector may bemore judiciously chosen, and (2) the practitioner will be directed torelated or similar species for further screening if a protein ofinterest is identified.

[0106] Compared to traditional fungal hosts, transformation, expressionand secretion rates are exceedingly high when using a Chrysosporiumstrain exhibiting the compact mycelial morphology of strain UV18-25.Thus a recombinant strain according to the invention will preferablyexhibit such morphology. The invention however also coversnon-recombinant strains or otherwise engineered strains of fungiexhibiting this characteristic. An attractive embodiment of theinvention would employ a recombinant Chrysosporium strain exhibiting aviscosity below that of strain NG7C- 19, preferably below that ofUV18-25 under corresponding or identical culture conditions. We havedetermined that the viscosity of a culture of UV18-25 is below 10 cP asopposed to that of previously known Trichoderma reesei being of theorder 200-600 cP, and with that of traditional Aspergillus niger beingof the order 1500-2000 cP under optimal culture conditions during themiddle to late stages of fermentation. Accordingly the invention mayemploy any engineered or mutant filamentous fungus exhibiting thislow-viscosity charactersistic, such as the Chrysosporium UV18-25 (VKMF-3631 D) strain, the Trichoderma X 252 strain, or A. sojae pclA(derived from ATCC 11906) or A. niger pclA.

[0107] The fluidity of filamentous fungal cultures can vary over a widerange, from nearly solid to a free-flowing liquid. Viscosity can readilybe quantitated by Brookfield rotational viscometry, use of kinematicviscosity tubes, falling ball viscometer or cup type viscometer.Fermentation broths are non-Newtonian fluids, and the apparent viscositywill be dependent to some extent upon the shear rate (Goudar et al.,Appl. Microbiol. Biotechnol. 1999 51:310-315). This effect is howevermuch less pronounced for the low-viscosity cultures employed in thepresent invention.

[0108] The use of such low viscosity cultures in the screening of anexpression library according to the method of the invention is highlyadvantageous. The screening of DNA libraries expressed in filamentousfungi has heretofore been limited to relatively slow and laboriousmethods. In general, once fungi have been transformed (and thetransformants optionally selected for), it has been necessary to preparespores or conidia, or to mechanically disrupt the mycelia, in order todisperse the library of transformed fungi into individual organisms orreproductive elements. This dispersal is necessary so that the separatedorganisms can be cultured into clonal colonies or cultures. The spores,conidia, or mycelial fragments are then diluted and “plated out” instandard culture dishes, and the individual colonies are inspected forcolor, alterations to the substrate, or other detectable indication ofthe presence of the protein activity or property being sought. Inanother approach, secreted proteins are blotted from the colonies onto amembrane, and the membrane is probed or examined for an indication ofthe presence of the protein activity or property of interest. Use ofmembranes has proved useful where proteolytic degradation of exogenousprotein is a problem (Asgeirsdottir et al., Appl. Environ. Microbiol.1999, 65:2250-2252). Such procedures are labor-intensive and have notproven amenable to automation, and as a result high-throughput screeningof fungally-expressed proteins has not heretofore been accomplished withconventional filamentous fungi. For purposes of this disclosure,high-throughput screening refers to any partially- or fully-automatedscreening method that is capable of evaluating the proteins expressed byabout 1,000 or more transformants per day, and particularly to thosemethods capable of evaluationg 5,000 or more transformants per day, andmost particularly to methods capable of evaluating 10,000 or moretransformants per day.

[0109] The automated high-throughput screening of a library oftransformed fungi according to the present invention, accordingly, maybe carried out in a number of known ways. Methods that are known to beapplicable to bacteria or yeast may in general be applied to thelow-viscosity fungi of the present invention. This is made possible bythe presence of transferable reproductive elements in combination withthe low-viscosity phenotype, a consequence of the relativelynon-entangled morphology of the hyphae of the mutant fungi employed. Inessence, the mutant fungi, and/or their transferable reproductiveelements, behave very much like individual bacteria or yeast during themechanical manipulations involved in automated high-throughputscreening. This is in contrast to wild-type fungi, and mostindustrially-adapted fungi as well, which produce highly entangledmycelia which do not permit the ready separation of the individualorganisms from one another.

[0110] For example, a dilute suspension of transformed fungi accordingto the present invention may be aliquotted out through a mechanicalmicropipette into the wells of a 96-well microplate. It is anticipatedthat liquid-handling apparatus capable of pipetting into 384- or1536-well microplates can also be adapted to the task of automateddispersal of the organisms into microplates. The concentration of thesuspended organisms can be adjusted as desired to control the averagenumber of organisms (or other transferable reproductive elements) perwell. It will be appreciated that where multiple individual organismsare aliquotted into wells, the identification of the desired proteinactivity or property in that well will be followed by dilution of thecontents of the well and culturing the organisms present into individualclonal colonies or cultures. In this manner the throughput of the systemmay be increased, at the cost of the need for subsequent resolution ofthe contents of each well that presents a “hit”.

[0111] In an alternative embodiment, a cell sorter may be interposed inthe fluid path, which is capable of directing the flow of the culture tothe wells of the microplate upon the detection of an organism or othertransferable reproductive element in the detector cell. This embodimentpermits the reasonably accurate dispensation of one organism per well.The use of an optically-detectable marker, such as green fluorescentprotein, to identify transformats is particularly useful in thisembodiment, as it permits the automated selection of transformants by afluorescence-activated cell sorter.

[0112] In yet another embodiment, colonies growing on solid media can bepicked by a robotic colony picker, and the organisms transferred by therobot to the wells of a microtiter plate. Well-separated colonies willgive rise to single clones in each well.

[0113] The dispersed organisms are then permitted to grow into clonalcultures in the microplate wells. Inducers, nutrients, etc. may be addedas desired by the automated fluid dispensing system. The system may alsobe used to add any reagents required to enable the detection of theprotein activity or property of interest. For example, colorogenic orfluorogenic substrates can be added so as to permit the spectroscopic orfluorometric detection of an enzyme activity. The low viscosity andsubmerged growth habit of the cultures in the wells of a microtiterplate permit the rapid diffusion of such reagents into the culture,greatly enhancing the sensitivity and reliability of the assay.Diffusion of oxygen and nutrients is also greatly enhanced, facilitatingrapid growth and maximal expression and secretion of exogenous peptides.Certain assays, such as the scintillation proximity assay, rely on thediffusion of soluble components so as to arrive at an equilibrium state;again the low viscosity of the fungal cultures of the present inventionmakes this high throughput assay possible. Finally, in a highlyautomated system it will be desirable to automatically pick, aspirate,or pipette clonal cultures of interest from their wells in themicrotiter plate, and the low viscosity and submerged growth habit ofthe cultures will make this possible. All of the above operations wouldbe difficult or impossible given the viscosity of traditional filamenousfungal cultures, especially cultures growing as surface mats in theunstirred, shear-free conditions of a microtiter plate well.

[0114] In another emodiment, single cells are passed through amicrofluidic apparatus, and the property or activity of interest isdetected optically (Wada et al., WO 99/67639). Low viscosity isessential to the operation of a microfluidics device, and cultures ofthe low-viscosity mutant fungi of the present invention are expected tobe amenable to microfluidic manipulation. Short et al., in U.S. Pat. No.6,174,673, have described how fluorogenic substrates may be employed todetect an enzyme activity of interest, and how host cells expressingsuch an activity may be isolated with a fluorescence-activated cellsorter. The methods of the present invention are compatible with thismethod of identification of expressed proteins.

[0115] In one embodiment, where transformants carry a fluorescentprotein as a marker, the fluorescence may be quantitated and employed asa measure of the amount of gene expression and/or expressed proteinpresent in a given culture. In this embodiment, it is possible not onlyto detect an exogenous protein of interest, but to estimate the specificactivity of the protein, as described by Blyna et al. in WO 00/78997.This embodiment will be particularly preferred where the screeningmethod of the invention is employed as part of a process of directedevolution.

[0116] In those cases where a greater viscosity is acceptable, agel-forming matrix may provide certain advantages when culturing fungi,and conducting biochemical assays, in a microplate format, as describedby Bochner in U.S. Pat. No. 6,046,021.

[0117] Another class of high-thoughput screens is by photometricanalysis, by digital imaging spectroscopy, of large numbers ofindividual colonies growing on a solid substrate. See for example Youvanet al., 1994, Meth. Enzymol. 246:732-748. In this method, changes in theoverall absorption or emission spectra of specialized reagents areindicative of the presence of a heterologous protein activity orproperty of interest. The ready dispersal of individual organismsattendant upon the use of low-viscosity mutants also enables the use offilamentous fungi in this method. The tendency for colonies of themutant fungi of the invention to exhibit less lateral growth, and toproduce smooth, compact, and well-defined colonies on solid media, isalso advantageous in such a screening system. Furthermore, the superiorexpression and secretion characteristics of fungi as compared tobacteria provide greater quantities of protein for spectral analysis.

[0118] An automated microorganism handling tool is described in Japanesepatent application publication number 11-304666. This device is capableof the transfer of microdroplets containing individual cells, and it isanticipated that the fungal strains of the present invention, by virtueof their morphology, will be amenable to micromanipulation of individualclones with this device.

[0119] An automated microbiological high-throughput screening system isdescribed in Beydon et al., J. Biomol. Screening 5:13-21 (2000). Therobotic system is capable of transferring droplets with a volume of 400nl to agar plates, and processing 10,000 screening points per hour, andhas been used to conduct yeast two-hybrid screens. It is anticipatedthat the fungal hosts of the present invention will be as amenable asyeast to high-throughput screening with systems of this type.

[0120] As an alternative to microtiter plates, transformants can begrown on plates and, in the form of microcolonies, assayed optically asdescribed in WO 00/78997.

[0121] The development of high throughput screens in general isdiscussed by Jayawickreme and Kost, Curr. Opin. Biotechnol. 8:629-634(1997). A high throughput screen for rarely transcribed differentiallyexpressed genes is described in von Stein et al., Nucleic Acids Res.35:2598-2602 (1997).

[0122] The Chrysosporium strain UV18-25 and the Trichoderma strain X 252illustrate various aspects of the invention exceedingly well. Theinvention however may employ other mutant or otherwise engineeredstrains of filamentous fungi that produce transferable reproductiveelements in suspension and exhibit low viscocity in culture. Thespecific morphology of the fungi may not be critical; the presentinventors have observed short, non-entangled mycelia in these twostrains but other morphologies, such as close and extensive hyphalbranching, may also lead to reduced viscosity. Fungal strains accordingto the invention are preferred if they exhibit optimal growth conditionsat neutral pH and temperatures of 25-43° C. Such screening conditionsare advantageous for maintaining the activity of exogenous proteins, inparticular those susceptible to degradation or inactivation at acidicpH. Most mammalian proteins, and human proteins in particular, haveevolved to function at physiological pH and temperature, and screeningfor the normal activity of a human enzyme is best carried out underthose conditions. Proteins intended for therapeutic use will have tofunction under such conditions, which also makes these the preferredscreening conditions. Chrysosporium strains exhibit precisely thischaracteristic, growing well at neutral pH and 35-40° C., while othercommonly employed fungal host species (e.g. Aspergillus and Trichoderma)grow best at acidic pH and may be less suitable for this reason.

[0123] Another application of the method of the present invention is inthe process of “directed evolution,” wherein novel protein-encoding DNAsequences are generated, the encoded proteins are expressed in a hostcell, and those seqences encoding proteins exhibiting a desiredcharacteristic are selected, mutated, and expressed again. The processis repeated for a number of cycles until a protein with the desiredcharacteristics is obtained. Gene shuffling, protein engineering,error-prone PCR, site-directed mutagenesis, and combinatorial and randommutagenesis are examples of processes through which novel DNA sequencesencoding exogenous proteins can be generated. U.S. Pat. Nos. 5,223,409,5,780,279 and 5,770,356 provide teaching of directed evolution. See alsoKuchner and Arnold, Trends in Biotechnology, 15:523-530 (1997);Schmidt-Dannert and Arnold, Trends in Biotech., 17:135-136 (1999);Arnold and Volkov, Curr. Opin. Chem. Biol., 3:54-59 (1999); Zhao et al.,Manual of Industrial Microbiology and Biotechnology, 2^(nd) Ed., (Demainand Davies, eds.) pp. 597-604, ASM Press, Washington D.C., 1999; Arnoldand Wintrode, Encyclopedia of Bioprocess Technology: Fermentation,Biocatalysis, and Bioseparation, (Flickinger and Drew, eds.) pp.971-987, John Wiley & Sons, New York, 1999; and Minshull and Stemmer,Curr. Opin. Chem. Biol. 3:284-290.

[0124] An application of combinatorial mutagenesis is disclosed in Hu etal., Biochemistry. 1998 37:10006-10015. U.S. Pat. No. 5,763,192describes a process for obtaining novel protein-encoding DNA sequencesby stochastically generating synthetic sequences, introducing them intoa host, and selecting host cells with the desired characteristic.Methods for effecting artificial gene recombination (DNA shuffling)include random priming recombination (Z. Shao, et al., Nucleic AcidsRes., 26:681-683 (1998)), the staggered extension process (H. Zhao etal., Nature Biotech., 16:258-262 (1998)), and heteroduplex recombination(A. Volkov et al., Nucleic Acids Res., 27:e18 (1999)). Error-prone PCRis yet another approach (Song and Rhee, Appl. Environ. Microbiol.66:890-894 (2000)).

[0125] There are two widely-practiced methods of carrying out theselection step in a directed evolution process. In one method, theprotein activity of interest is somehow made essential to the survivalof the host cells. For example, if the activity desired is a cellulaseactive at pH 8, a cellulase gene could be mutated and introduced intothe host cells. The transformants are grown with cellulose as the solecarbon source, and the pH raised gradually until only a few survivorsremain. The mutated cellulase gene from the survivors, which presumablyencodes a cellulase active at relatively high pH, is subjected toanother round of mutation, and the process is repeated untiltransformants that can grow well on cellulose at pH 8 are obtained.Thermostable variants of enzymes can likewise be evolved, by cycles ofgene mutation and high-temperature culturing of host cells (Liao et al.,Proc. Natl. Acad. Sci. USA 1986 83:576-580; Giver et al., Proc. Natl.Acad. Sci. USA. 1998 95:12809-12813. For purposes of this application,mutation of DNA sequences encoding exogenous proteins may beaccomplished by any of several methods employed for directed evolution,for example by gene shuffling, in vivo recombination, or cassettemutagenesis.

[0126] The chief advantage of this method is the massively parallelnature of the “survival of the fittest” selection step. Millions, orbillions, of unsuccessful mutations are simultaneously eliminated fromconsideration without the need to evaluate them individually. However,it is not always possible to link an enzyme activity of interest to thesurvival of the host. For example where the desired protein property isselective binding to a target of interest, making the binding propertyessential to survival is likely to be difficult. Also, survival underforced conditions such as high temperature or extreme pH is likely to bedependent upon multiple factors, and a desirable mutation will not beselected for and will be lost if the host cell is unable to survive forreasons unrelated to the properties of the mutant protein.

[0127] An alternative to the massively parallel “survival of thefittest” approach is serial screening. In this approach, individualtransformants are screened by traditional methods, such as observationof cleared or colored zones around colonies growing on indicator media,colorimetric or fluorometric enzyme assays, immunoassays, bindingassays, etc. See for example Joo et al., Nature 399:670-673 (1999),where a cytochrome P450 monooxygenase not requiring NADH as a cofactorwas evolved by cycles of mutation and screening; May et al., NatureBiotech. 18:317-320 (2000), where a hydantoinase of reversedstereoselectivity was evolved in a similar fashion; and Miyazaki et al.,J. Mol. Biol. 297:1015-1026 (2000), where a thermostable subtilisin wasevolved.

[0128] The screening approach has clear advantages over a simple“survival screen,” especially if it can be carried out in ahigh-throughput manner that approaches the throughput of the massivelyparallel “survival screen” technique. For example, a degree ofparallelism has been introduced by employing such measures as digitalimaging of the transformed organisms (Joo et al., Chemistry & Biology,6:699-706 (1999)) or digital spectroscopic evaluation of colonies(Youvan et al., 1994, Meth. Enzymol. 246:732-748). Serial assays can beautomated by the use of cell sorting (Fu et al., Nature Biotech.,17:1109-1111 (1999)). A well-established approach to high-throughputscreening involves the automated evaluation of expressed proteins inmicrotiter plates, using commercially available plate readers, and themethod of the present invention is well-suited to the application ofthis mode of high-throughput screening to directed evolution.

[0129] In this embodiment of the invention, a gene encoding a protein ofinterest is mutated by any known method of generating a plurality ofmutants, the mutant protein-encoding DNA is introduced by means of asuitable expression vector into a low-viscosity filamentous fungal hostaccording to the present invention, and the transformants are optionallyselected for and cultured. The host cells are then dispersed asdescribed previously into the wells of a microtiter plate, or otherwisespatially separated into resolvable locations, so as to provideindividual monoclonal cultures (or poly-clonal cultures having fewerthan about 100 different clones). The cells are preferably dispersedinto the wells of a micro-titer plate. The protein encoded by the mutantDNA is preferably secreted into the medium in the wells of themicrotiter plates. Each of the dispersed cultures is screened for theprotein activity of interest, and those most strongly exhibiting thedesired property are selected. The gene encoding the protein of interestin the selected cultures is mutated again, the mutant DNA is againintroduced into the low-viscosity fungal host, and the transformants arere-screened. The mutating and re-screening process is repeated until thevalue of the property of interest reaches a desired level.

[0130] In an alternative embodiment, directed evolution is carried outby mutation and reproduction of the gene of interest in anotherorganism, such as E. coli, followed by transfer of the mutant genes to afilamentous fungus according to the present invention for screening.

[0131] It will be readily appreciated by those skilled in the art that aprotein that appears to be of interest based upon the screening assaywill not necessarily have all the other properties required forcommercial utility. For example, the possession of enzymatic activity,however high the specific activity, will not indicate that the mutantenzyme has the requisite thermal or pH stability, or detergent orprotease resistance, or non-immunogenicity, or other property that mightbe desirable or necessary in a commercially viable product. There is aneed for methods of readily determining whether an identified proteinhas commercially useful properties.

[0132] The prior art approaches to screening have not provided asolution to this need, because the host organisms (bacteria and yeast)were not adapted to the production of isolable quantities of protein. Ithas heretofore been necessary to transfer potentially useful genes fromone organism to another, as one proceeded through DNA librarypreparation, gene expression, screening, expression of researchquantities of gene products, and over-expression in industriallysuitable production strains. The mutant filamentous fungi of the presentinvention, on the other hand, are excellent overproducers and secretorsof exogenous proteins, especially when employed with the vectorsdisclosed herein. Sufficient protein may be isolated not only forpurposes of characterization, but for evaluation in application trials.Indeed, the strains used in the screening method of the invention aresuitable for industrial production as well, since they possess desirableproduction properties such as low viscosity, high expression rates, andvery high protein/biomass ratios.

[0133] Accordingly, in a preferred embodiment of the present invention,the method further comprises culturing a clonal colony or cultureidentified according to the method of the invention, under conditionspermitting expression and secretion of the exogenous library protein (ora precursor thereof), and recovering the subsequently produced proteinto obtain the protein of interest. Expression and secretion of a libraryprotein may be facilitated by creating an in-frame fusion of the clonedgene with the gene for a heterologous protein (or a fragment thereof)with its corresponding signal sequence, or with the signal sequence froma third protein, all operably linked to an expression regulatingsequence. By this approach a fusion protein is created that containsheterologous amino acid sequences upstream of the library protein.Subsequently, this fusion precursor protein may be isolated andrecovered using purifaction techniques known in the art. The method mayoptionally comprise subjecting the secreted fusion protein precursor toa cleavage step to generate the library protein of interest. Thecleavage step can be carried out with Kex-2, a Kex-2 like protease, oranother selective protease, when the vector is engineered so that aprotease cleavage site links a well-secreted protein carrier and theprotein of interest.

[0134] The ready availability of mutant protein, directly from thescreening host organism, has not previously been possible with prior artscreening hosts. The present invention thus provides an advantage, inthat the mutant proteins deemed of interest based upon thehigh-throughput screen can be isolated in sufficient quantities(milligrams) for further characterization and even larger quantities(grams to kilograms) for application trials. This particular embodimentof the invention thus permits the practitioner to select mutant proteinsfor the next round of directed evolution based upon any number ofdesirable properties, and not merely upon the one property detected inthe high-throughput screen. The more stringent selection criteria madepossible by the present invention should lead to a more efficient andcost-effective directed evolution process.

[0135] The method of production of a recombinant mutant filamentousfungal strain according to the invention comprises introducing a libraryof DNA sequences comprising nucleic acid sequences encoding heterologousproteins into a low-viscosity mutant filamentous fungus according to theinvention, the nucleic acid sequences being operably linked to anexpression regulating region. The introduction of the DNA sequences maybe carried out in any manner known per se for transforming filamentousfungi. Those skilled in the art will appreciate that there are severalwell-established methods, such as CaCl₂-polyethylene glycol stimulatedDNA uptake by fungal protoplasts (Johnstone et al., EMBO J., 1985,4:1307-1311). A protoplast transformation method is described in theexamples. Alternative protoplast or spheroplast transformation methodsare known and can be used as have been described in the prior art forother filamentous fungi. Vectors suitable for multicopy integration ofheterologous DNA into the fungal genome are well-known; see for exampleGiuseppin et al., WO 91/00920. The use of autonomously replicatingplasmids has long been known as an efficient transformation tool forfungi (Gems et al., Gene 1991 98:61-67; Verdoes et al., Gene 1994146:159-165; Aleksenko and Clutterbuck, Fungal Genetics Biol. 199721:373-387; Aleksenko et al., Mol. Gen. Genet. 1996 253:242-246).Details of such methods can be found in many of the cited references,and they are thus incorporated by reference.

[0136] Exemplary methods according to the invention, comprising using alow-viscosity mutant strain of Chrysosporium or A. sojae as startingmaterial for introduction of vectors carrying heterologous DNA, arepresented below.

EXAMPLES

[0137] A. Development of Compact Growth Morphology Mutants

[0138] Various patent applications teach that morphological mutants canbe isolated by various ways of screening. WO 96/02653 and WO 97/26330describe non-defined mutants exhibiting compact morphology. It was foundthat a proprotein processing mutant of A. sojae had an unexpectedaberrant growth phenotype (hyper-branching) while no detrimental effecton protein production were observed. Culture experiments with thisstrain revealed a very compact growth phenotype with micropellets. Theobserved characteristics were not only present in A. sojae but othermutated fungi as well, e.g. A. niger.

[0139] (1) Construction of an A. niger Proprotein Processing Mutant

[0140] To clone the proprotein convertase encoding gene from A. niger,PCR was used. Based on the comparison of various proprotein convertasegenes from various yeast species and higher eukaryotes, different PCRprimers were designed which are degenerated, respectively, 4, 2, 2, 512,1152, 4608, 2048 and 49152 times. From the amplification using primersPE4 and PE6, two individual clones were obtained of which the encodedprotein sequence did show significant homology to the S. cerevisiae KEX2sequence. These clones were used for further experiments.

[0141] Based on the observed homology to other proprotein convertasegenes of the cloned PCR fragment, the corresponding A. niger gene wasdesignated pclA (from proprotein-convertase-like). Southern analysis ofgenomic digests of A. niger revealed that the pclA gene was a singlecopy gene with no closely related genes in the A. niger genome, as evenat heterologous hybridisation conditions (50° C.; washes at 6×SSC) noadditional hybridisation signals were evident. A first screening of anEMBL3 genomic library of A. niger N401 (van Hartingsveldt et al., Mol.Gen. Genet 1987 206:71-75) did not result in any positively hybridisingplaques although about 10-20 genome equivalents were screened. In asecond screening a fall length genomic copy of the pclA gene wasisolated from an A. niger N400 genomic library in EMBL4 (Goosen et al.,Curr. Genet. 11:499-503 (1987)).

[0142] Of the 8 hybridising plaques which were obtained after screening5-10 genome equivalents, 6 were still positive after a firstrescreening. All these 6 clones most likely carried a full copy of thepclA gene, as in all clones (as was observed for the genomic DNA) withthe PCR fragment two hybridising EcoRV fragments of 3 and 4 kb werepresent (the PCR fragment contained an EcoRV restriction site). Based oncomparison of the size of other proprotein convertases, together thesefragments will contain the complete pclA gene with 5′ and 3′ flankingsequences. The two EcoRV fragments and an overlapping 5 kb EcoRIfragment were subcloned for further characterisation.

[0143] Based on the restriction map the complete DNA sequence of thepclA gene was determined from the EcoRI and EcoRV subclones. Analysis ofthe obtained sequence revealed an open reading frame with considerablesimilarity to that of the S. cerevisiae KEX2 gene and other proproteinconvertases. Based on further comparison two putative intron sequenceswere identified in the coding region. Subsequent PCR analysis withprimers flanking the putative introns, on a pEMBLyex based A. niger cDNAlibrary revealed that only the most 5′ of these two sequencesrepresented an actual intron. The general structure of the encoded PclAprotein was clearly similar to that of other proprotein convertases. Theoverall similarity of the PclA protein with the other proproteinconvertases was about 50%.

[0144] To demonstrate that the cloned pclA gene is a functional geneencoding a functional protein, the construction of strains devoid of thepclA gene was attempted. Therefore, pPCL1A, a pclA deletion vector, inwhich a large part of the pclA coding region was replaced for the A.oryzae pyrG selection marker, was generated. Subsequently, from thisvector the 5 kb EcoRI insert fragment was used for transformation ofvarious A. niger strains.

[0145] From these transformations (based on pyrG selection) numeroustransformants were obtained. Interestingly, a fraction of thetransformants (varying from 1-50%) displayed a very distinct aberrantphenotype (FIG. 13). Southern analysis of several wildtype and aberranttransformants revealed that these aberrant transformants which displayeda severely restricted (compact) growth phenotype, had lost the pclAgene. All strains displaying wild-type growth were shown to carry a copyof the replacement fragment integrated adjacent to the wild-type pclAgene or at a non-homologous position.

[0146] (2) Construction of an A. sojae Proprotein Processing Mutant

[0147] To construct the corresponding mutant in A. sojae, functionalcomplementation of the low-viscosity mutant of A. niger was carried outby transformation of an A. niger pclA mutant with the A. sojae ATCC11906 cosmid library. From the resulting complemented A. nigertransformants, genomic cosmid clones were isolated, which comprised theA. sojae protein processing protease pclA. Partial sequence analysis ofthe isolated sequences confirmed the cloning of the A. sojae pclA gene.Based on the cloned A. sojae pclA sequences a gene replacement vectorwas generated following an approach similar to that described elsewherein our examples, using the reusable pyrG selection marker described inWO 01/09352.

[0148] In addition, a gene disruption vector was constructed carryingthe pyrG selection marker and 5′ and 3′ truncated fragment from the A.sojae pclA gene. Both the gene replacement and gene disruption vectorwere used to generate pclA mutants in ATCC 11906 and ATCC 11906derivatives. Culture experiments with some of the resultingtransformants revealed improved morphological characteristics, inparticular compact growth morphology and micropellets. (FIGS. 14A and14B)

[0149] (3) Isolation of Alternative A. sojae Compact Growth Mutants

[0150] Transformation of A. sojae ATCC 11906 and derivatives may becarried out with linear DNA fragments carrying a fungal selectionmarker. If no specific replicating sequences are provided transformantsobtained using this procedure carry the introduced DNA integrated intothe genome of the host strain. As the introduced selection marker isfrom heterologous origin (A. niger) only heterologous recombination willoccur, leading to a collection of transformants carrying the marker DNAat various positions in the genome. This integration is prone to resultin disruption of endogenous A. sojae sequences, thus resulting in acollection of A. sojae mutant strains. This is exemplified by theanalysis of a large collection of transformants obtained from A. sojaeATCC 1906alpApyrG using a DNA fragment with the A. niger pyrG selectionmarker. In total several thousand transformants were analysed and fromthese 5-10 showed a morphologically aberrant phenotype. Amoung theseseveral had a phenotype comparable to the pclA mutants. Similar asdescribed for the cloning of the A. sojae pclA gene, the genecorresponding to the mutation could be isolated from the A. sojae genelibrary by complementation of the morphological phenotype. Based on thecloned gene the corresponding gene disruption/deletion mutants can begenerated.

[0151] (4) Isolation of Chrysosporium Compact Growth Mutants.

[0152] Using a similar PCR based cloning approach as described for theA. niger pclA gene a fragment of the Chrysosporium proprotein processinggene, termed pcl1, was cloned from a Chrysosporium BLUESTAR (™) genelibrary. A gene fragment carrying the complete genomic gene copy wassubcloned from the pBLUESTAR clone. Based on the obtained subclone agene disruption vector was generated as described for A. sojae. Insteadof the pyrG marker, for Chrysosporium the repeat flanked version of theA. niger pyrE gene was used. Gene disruption-transformation ofChrysosporium resulted in strains with a compact growth phenotype.

[0153] B. Viscosity Determinations

[0154] The following operating parameter data ranges have beendetermined for fungal fermentations using five different fungalorganisms. The five fungal organisms compared were strains ofAspergillus niger, Trichoderma longibrachiatum 18.2KK (formerly T.reesei), Trichoderma longibrachiatum×252, Chrysosporium lucknowensestrain UV18-25, and Aspergillus sojae pclA. Viscosity of a fungalculture varies during the course of a fermentation, and varies withnutrient concentration. For the measurements reported here, mediumcontaining between 20 and 100 g/l of a carbohydrate carbon source (e.g.,cellulose, lactose, sucrose, xylose, glucose, and the like) isinoculated with the fungus, and the culture allowed to proceed through a“growth phase” during which the carbon source is consumed. Shake flaskcultures are shaken at 200 rpm, while one-liter fermentation vessels arestirred with an impeller at 500-1000 rpm. Maximal viscosity typicallyoccurs at or close to the end of the growth phase. At this time theculture is switched to a fed batch mode, wherein a carbon source is fedto the culture at a rate such that the concentration of the carbonsource does not rise above about 0.5 g/l. A feed rate of between 1 and 3g/l/hr is typical.

[0155] Viscosity was determined on a Brookfield LVF viscometer using thesmall sample adapter and spindle number 31, operated at 30° C. A freshsample of fermentation broth (10 ml) was placed in the small samplespindle. The spindle speed was adjusted to give a reading in the range10-80. After four minutes a reading was taken from the viscometer scale.The reading was multiplied by the factor given below to get theviscosity in centipoise (cP). Spindle Speed Multiplication Factor  6 5012 25 30 10 60  5

[0156] The final viscosity was measured at fermentation end: StrainFinal viscosity, cP (mean ± s.d.) T. longibrachiatum 18.2KK (297 ± 173)A. niger 1,500-2,000 T longibrachiatum X-252 ≦60 C. lucknowense UV18-25≦10 A. sojae pclA n.d.

[0157] C. Transformation of Chrysosporium, Trichoderma and Tolypocladium

[0158] Transformation media used were as follows: Mandels Base: MnPMedium: KH₂PO₄ 2.0 g/l Mandels Base with (NH₄)₂SO₄ 1.4 g/l Peptone 1 g/lMgSO₄.7H₂O 0.3 g/l MES 2 g/l CaCl₂ 0.3 g/l Sucrose 100 g/l Oligoelements1.0 ml/l Adjust pH to 5 MnR MnP Ca²⁺: MnP + sucrose 130 g/l MnP Medium +Yeast extract 2.5 g/l CaCl₂ 2H₂O, 50 mM Glucose 2.5 g/l Adjust pH to 6.5Agar 15 g/l MnR Soft: MnR with only 7.5 g/l of agar. MPC: CaCl₂ 50 mM pH5.8 MOPS 10 mM PEG 40% Media for selection and culture: GS: Glucose 10g/l Biosoyase 5 g/l [Merieux] Agar 15 g/l pH should be 6.8 PDA: PotatoDextrose Agar (Difco) 39 g/l pH should be 5.5 MPG: Mandels Base with 5g/l K Phtalate Glucose 30 g/l Yeast extract 5 g/l IC1  0.5 g/L K2HPO4 pH7.0  0.15 g/L MgSO4.7H20  0.05 g/L KCl 0.007 g/L FeSO4.7H2O    1 g/LYeast extract (ohly KAT)   10 g/L Peptone or Pharmamedia   10 g/Llactose   10 g/L glucose

[0159] The regeneration media (MnR) supplemented with 50 μg/mlphleomycin or 100-150 μg/ml hygromycin is used to select transformants.GS medium, supplemented with 5 μg/ml phleomycin is used to confirmantibiotic resistance.

[0160] PDA is a complete medium for fast growth and good sporulation.Liquid media are inoculated with {fraction (1/20)}th of spore suspension(all spores from one 90 mm PDA plate in 5 ml 0.1% Tween). Such culturesare grown at 27° C. in shake flasks (200 rpm).

[0161] Two untransformed Chrysosporium C1 strains and one Trichodermareesei reference strain were tested on two media (GS pH 6.8, and Pridhamagar, PA, pH 6.8). To test the antibiotic resistance level spores werecollected from 7 day old PDA plates. Selective plates were incubated at32° C. and scored after 2, 4 and 5 days. The C-1 strains NG7C-19 andUV18-25 clearly had a low basal resistance level both to phleomycin andhygromycin, comparable to that for a reference T. reesei laboratorystrain. This is a clear indication these standard fungal selectablemarkers can be used in Chrysosporium strains. Problems with otherstandard fungal selectable markers are not expected.

[0162] Selection of Sh-ble (phleomycin-resistance) transformedChrysosporium strains was successfully carried out at 50 μg/ml. This wasalso the selection level used for T. reesei thus showing thatdifferential selection can be easily achieved in Chrysosporium. The samecomments are valid for strains transformed for hygromycin resistance ata level of 150 μg/ml.

[0163] The protoplast transformation technique was used on Chrysosporiumbased on the most generally applied fungal transformation technology.All spores from one 90 mm PDA plate were recovered in 8 ml IC1 andtransferred into a shake flask of 50 ml IC1 medium for incubation for 15hours at 35° C. and 200 rpm. After this the culture was centrifuged, thepellet was washed in MnP, brought back into solution in 10 ml MnP and 10mg/ml Caylase C₃ and incubated for 30 minutes at 35° C. with agitation(150 rpm).

[0164] The solution was filtered and the filtrate was subjected tocentrifugation for 10 minutes at 3500 rpm. The pellet was washed with 10ml MnP Ca²⁺. This was centrifuged for 10 minutes at 25° C. Then 50microlitres of cold MPC was added. The mixture was kept on ice for 30minutes whereupon 2.5 ml PMC was added. After 15 minutes at roomtemperature 500 microlitres of the treated protoplasts were mixed to 3ml of MnR Soft and immediately plated out on a MnR plate containingphleomycin or hygromycin as selection agent. After incubation for fivedays at 30° C. transformants were analysed (clones become visible after48 hours). Transformation efficiency was determined using 10 μg ofreference plasmid pAN8-1. The results are presented in the followingTable C. TABLE C Transformation efficiency (using 10 μg of referenceplasmid pAN8-1) T. reesei NG7C-19 UV18-25 Viability  10⁶/200 μl   5 ×10⁶/200 μl   5 × 10⁶/200 μl Transformants 2500  10⁴  10⁴ Per 200 μlTransformants per 2500 2000 2000 10⁶ viable cells

[0165] The results show that the Chrysosporium transformant viability issuperior to that of Trichoderma. The transformability of the strains iscomparable and thus the number of transfornants obtained in oneexperiment lies 4 times higher for Chrysosporium than for T. reesei.Thus the Chrysosporium transformation system not only equals thecommonly used T. reesei system, but even outperforms it. Thisimprovement can prove especially useful for vectors that are lesstransformation efficient than pAN8-1.

[0166] A number of other transformation and expression plasmids wereconstructed with homologous Chrysosporium protein encoding sequences andalso with heterologous protein encoding sequences for use intransformation experiments with Chrysosporium. The vector maps areprovided in FIGS. 6-11.

[0167] The homologous protein to be expressed was selected from thegroup of cellulases produced by Chrysosporium and consisted ofendoglucanase 6 which belongs to family 6 (MW 43 kDa) and theheterologous protein was endoglucanase 3 which belongs to family 12 (MW25 kDa) of Penicillium.

[0168] pF6 g comprises Chrysosporium endoglucanase 6 promoter fragmentlinked to endoglucanase 6 signal sequence in frame with theendoglucanase 6 open reading frame followed by the endoglucanase 6terminator sequence. Transformant selection is carried out by usingcotransformation with a selectable vector.

[0169] pUT1150 comprises Trichoderma reesei cellobiohydrolase promoterlinked to endoglucanase 6 signal sequence in frame with theendoglucanase 6 open reading frame followed by the T. reeseicellobiohydrolase terminator sequence. In addition this vector carries asecond expression cassette with a selection marker, i.e. the phleomycinresistance gene (Sh-ble gene).

[0170] pUT1152 comprises Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase A promoter linked to endoglucanase 6 signal sequence inframe with the endoglucanase 6 open reading frame followed by the A.nidulans anthranilate synthase (trpC) terminator sequence. In additionthis vector carries a second expression cassette with a selectionmarker, i.e. the phleomycin resistance gene (Sh-ble gene).

[0171] pUT1155 comprises A. nidulans glyceraldehyde-3-phosphatedehydrogenase A promoter linked to Trichoderma reesei cellobiohydrolasesignal sequence in frame with the carrier protein Sh-ble which in turnis linked in frame to the endoglucanase 6 open reading frame followed bythe A. nidulans trpC terminator sequence. This vector uses thetechnology of the carrier protein fused to the protein of interest whichis known to very much improve the secretion of the protein of interest.

[0172] pUT1160 comprises Aspergillus nidulans glyceraldehyde-3-phosphatedehydrogenase A promoter linked to Trichoderma reesei cellobiohydrolasesignal sequence in frame with the carrier protein Sh-ble which in turnis linked in frame to the endoglucanase 3 open reading frame ofPenicillium followed by the A. nidulans trpC terminator sequence.

[0173] pUT1162 comprises Trichoderma reesei cellobiohydrolase promoterlinked to endoglucanase 3 signal sequence in frame with theendoglucanase 3 open reading frame of Penicillium followed by the T.reesei cellobiohydrolase terminator sequence. In addition this vectorcarries a second expression cassette with the phleomycin resistance gene(Sh-ble gene) asa selection marker.

[0174] It will be apparent to those skilled in the art that a sample ofgenomic or cDNA can be readily sheared or digested into protein-encodingfragments, and the fragments ligated into vectors such as thoseillustrated herein so as to produce a library of expression vectors. Itwill be further apparent that methods employing co-transfection areapplicable, and that autonomously replicating vectors or integratingvectors may be employed to transfect filamentous fungi with such alibrary of vectors. TABLE D Comparative transformations Vector StrainTransformation No of transf. pUT1150 UV18-25 selection on phleomycin 285T. geodes selection on phleomycin 144 pUT1152 UV18-25 cotransformationpAN8.1 398 T. geodes cotransformation pAN8.1  45 pF6g UV18-25cotransformation pAN8.1 252 T. geodes cotransformation pAN8.1 127pUT1162 UV18-25 selection on phleomycin >400   T. geodes (n.d.)

[0175] Table D shows the results of transformation of both ChrysosporiumUV18-25 and Tolypocladium geodes. The transformation protocol used isdescribed below in the section for heterologous transformation.

[0176] D. Heterologous and Homologous Expression in ChrysosporiumTransformants

[0177] C1 strains (NG7C- 19 and/or UV18-25) were tested for theirability to secrete various heterologous proteins: a bacterial protein(Streptoalloteichus hindustanus phleomycin-resistance protein, Sh-ble),a fungal protein (Trichoderma reesei xylanase II, XYN2) and a humanprotein (the human lysozyme, HLZ). The details of the process are asfollows:

[0178] (1) C1 secretion of Streptoalloteichus hindustanusphleomycin-resistance protein (Sh-ble).

[0179] C1 strains NG7C-19 and UV18-25 were transformed by the plasmidpUT720 (ref. 1).

[0180] This vector presents the following fungal expression cassette:

[0181]Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase(gpdA) promoter (ref. 2)

[0182] A synthetic Trichoderma reesei cellobiohydrolase I (cbh1) signalsequence (refs 1, 3)

[0183]Streptoalloteichus hindustanus phleomycin-resistance gene Sh-ble(ref. 4)

[0184]Aspergillus nidulans tryptophan-synthase (trpC) terminator (ref.5)

[0185] The vector also carries the beta-lactamase gene (bla) and E. colireplication origin from plasmid pUC18 (Ref 6). The detailed plasmid mapis provided in FIG. 2.

[0186] C1 protoplasts were transformed according to Durand et al. (ref.7) adapted to C 1: All spores from one 90 mm PDA plate of untransformedC1 strain were recovered in 8 ml IC1 and transferred into a shake flaskwith 50 ml IC1 medium for incubation 15 hours at 35° C. and 150 rpm.Thereupon, the culture was spun down, the pellet washed in MnP, resolvedin 10 ml MnP+10 mg/ml Caylase C₃, and incubated 30 mm at 35° C. withagitation (150 rpm). The solution was filtered and the filtrate wascentrifuged 10 min at 3500 rpm. The pellet was washed with 10 mlMnPCa²⁺. This was spun down 10 min at 3500 rpm and the pellet was takenup into 1 ml MnPCa²⁺. 10 μg of pUT720 DNA were added to 200 μl ofprotoplast solution and incubated 10 min at room temperature (ca. 20°C.). Then, 50 μl of cold MPC was added. The mixture was kept on ice for30 min whereupon 2.5 ml PMC was added. After 15 min at room temperature500 μl of the treated protoplasts were mixed to 3 ml of MnR Soft andimmediately plated out on a MnR plate containing phleomycin (50 μg/ml atpH6.5) as selection agent. After 5 days incubation at 30° C.,transformants were analysed (clones start to be visible after 48 hours).

[0187] The Sh-ble production of C1 transformants (phleomycin-resistantclones) was analysed as follows: Primary transformants were toothpickedto GS+phleomycin (5 μg/ml) plates and grown for 5 days at 32° C. forresistance verification. Each validated resistant clone was subclonedonto GS plates. Two subclones per transformant were used to inoculatePDA plates in order to get spores for liquid culture initiation. Theliquid cultures in IC1 were grown 5 days at 27° C. (shaking 200 rpm).Then, the cultures were centrifuged (5000 g, 10 min.) and 500 μl ofsupernatant were collected. From these samples, the proteins wereprecipitated with TCA and resuspended in Western Sample Buffer to 4mg/ml of total proteins (Lowry method, Ref. 8). 10 μl (about 40 μg oftotal proteins) were loaded on a 12% acrylamide/SDS gel and run (MiniTrans-Blot™ system, BioRad Laboratories). Western blotting was conductedaccording to BioRad instructions (Schleicher & Schull 0.2 μm membrane)using rabbit anti-Sh-ble antiserum (Societe Cayla, Tolouse FR, Catalog#ANTI-0010) as primary antibody. The results are shown in FIG. 1 andTable E. TABLE E Sh-ble estimated production levels in C1 EstimatedSh-ble Estimated Sh-ble quantity on concentration in the the Westernblot production media Untransformed NG7C-19 Not detectable NG7C-19::720clone 4-1  25 ng 0.25 mg/l  NG7C-19::720 clone 5-1  25 ng 0.25 mg/l NG7C-19::720 clone 2-2 250 ng 2.5 mg/l Untransformed UV18-25 Notdetectable UV18-25::720 clone 1-2 500 ng 5.0 mg/l UV18-25::720 clone 3-1250 ng 2.5 mg/l

[0188] These data show that:

[0189] 1) The heterologous transcription/translation signals from pUT720are functional in Chrysosporium.

[0190] 2) The heterologous signal sequence of pUT720 is functional inChrysosporium.

[0191] 3) Chrysosporium can be used a host for the secretion ofheterologous bacterial proteins.

[0192] (2) C1 secretion of human lysozyme (HLZ).

[0193] C1 strains NG7C-19 and UV18-25 were transformed by the plasmidpUT970G (ref. 9).

[0194] This vector presents the following fungal expression cassette:

[0195]Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase(gpdA) promoter (ref. 2)

[0196] A synthetic Trichoderma reesei cellobiohydrolase I (cbh1 ) signalsequence (refs. 1, 3)

[0197]Streptoalloteichus hindustanus phleomycin-resistance gene Sh-ble 4used as carrier protein (ref. 10)

[0198]Aspergillus niger glucoamylase (glaA2) hinge domain cloned fromplasmid pAN56-2 (refs. 11, 12)

[0199] A linker peptide (LGERK) featuring a KEX2-like protease cleavagesite (ref. 1)

[0200] A synthetic human lysozyme gene (hlz) (ref 10)

[0201]Aspergillus nidulans tryptophan-synthase (trpC) terminator (ref.5)

[0202] The vector also carries the beta-lactamase gene (bla) and E. colireplication origin from plasmlid pUC18 6. The detailed plasmid map isprovided in FIG. 3.

[0203] C1 protoplasts were transformed with plasmid pUT970G followingthe same procedure already described in example 1. The fusion protein(Sh-ble: GAM hinge: HLZ) is functional with respect to thephleomycin-resistance thus allowing easy selection of the C1transformants. Moreover, the level of phleomycin resistance correlatesroughly with the level of hlz expression.

[0204] The HLZ production of C1 transformants (phleomycin-resistantclones) was analysed by lysozyme-activity assay as follow: Primarytransformants were toothpicked to GS+phleomycin (5 μg/ml) plates(resistance verification) and also on LYSO plates (HLZ activitydetection by clearing zone visualisation (refs. 1, 10). Plates weregrown for 5 days at 32° C. Each validated clone was subdloned onto LYSOplates. Two subdlones per transformant were used to inoculate PDA platesin order to get spores for liquid culture initiation. The liquidcultures in IC1 were grown 5 days at 27° C. (shaking 180 rpm). Then, thecultures were centrifuged (5000 g, 10 min.). From these samples,lysozyme activity was measured according to Mörsky et al. (ref. 13)TABLE F Active HLZ production levels in C1 Active HLZ concentration inculture media Untransformed NG7C-19 0 mg/l NG7C-19::970G clone 4 4 mg/lNG7C-19::970G clone 5 11 mg/l  Untransformed UV18-25 0 mg/lUV18-25::970G clone 1 8 mg/l UV18-25::970G clone 2 4 mg/l UV18-25::970Gclone 3 2 mg/l UV18-25::970G clone 2 2.5 mg/l  

[0205] These data show that:

[0206] 1) Points 1 & 2 from example 1 are confirmed.

[0207] 2) Sh-ble is functional in Chrysosporium as resistance marker.

[0208] 3) Sh-ble is functional in Chrysosporium as carrier protein.

[0209] 4) The KEX2-like protease cleavage site is functional inChrysosporium (otherwise HLZ would not be active).

[0210] 5) Chrysosporium can be used as host for the secretion ofheterologous mammalian proteins.

[0211] (3) C1 secretion of Trichoderma reesei xylanase II (XYN2).

[0212] C1 strain UV18-25 was transformed by the plasmids pUT1064 andpUT1065. pUT1064 presents the two following fungal expression cassettes:

[0213] The first cassette allows the selection of phleomycin-resistanttransformants:

[0214]Neurospora crassa cross-pathway control gene 1 (cpc-1) promoter(ref. 14)

[0215]Streptoalloteichus hindustanus phleomycin-resistance gene Sh-ble(ref. 4)

[0216]Aspergillus nidulans tryptophan-synthase (trpC) terminator (ref.5)

[0217] The second cassette is the xylanase production cassette:

[0218]T. reesei strain TR2 cbh1 promoter (ref. 15)

[0219]T. reesei strain TR2 xyn2 gene (including its signal sequence)(ref. 16)

[0220]T. reesei strain TR2 cbh1 terminator (ref. 15)

[0221] The vector also carries an E. coli replication origin fromplasmid pUC19 (ref. 6). The plasmid detailed map is provided in FIG. 4.

[0222] pUT1065 presents the following fungal expression cassette:

[0223]A. nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA)promoter (ref. 2)

[0224] A. synthetic T. reesei cellobiohydrolase I (cbh1) signal sequence(refs. 1, 3)

[0225]S. hindustanus phleomycin-resistance gene Sh-ble 4 used ascarrier-protein (ref. 10)

[0226] A linker peptide (SGERK) featuring a KEX2-like protease cleavagesite (ref. 1)

[0227]T. reesei strain TR2xyn2 gene (without signal sequence) (ref. 16)

[0228]A. nidulans tryptophan-synthase (trpC) terminator (ref. 5)

[0229] The vector also carries the beta-lactamase gene (bla) and an E.coli replication origin from plasmid pUC18 (Ref 6). The plasmid detailedmap is provided in FIG. 5.

[0230] C1 protoplasts were transformed with plasmid pUT1064 or pUT1065following the same procedure already described in example 1. The fusionprotein in plasmid pUT1065 (Sh-ble: XYN2) is functional with respect tothe phleomycin-resistance thus allowing easy selection of the C1transformants. Moreover, the level of phleomycin resistance correlatesroughly with the level of xyn2 expression. In pUT1064, xyn2 was clonedwith its own signal sequence.

[0231] The xylanase production of C1 transformants (phleomycin-resistantclones) was analysed by xylanase-activity assay as follow: Primarytransformants were toothpicked to GS+phleomycin (5 μg/ml) plates(resistance verification) and also on XYLAN plates (Ref. 17), wherexylanase activity is detected by observation of a clearing zone. Plateswere grown for 5 days at 32° C. Each validated clone was subdloned ontoXYLAN plates. Two subdlones per transformant were used to inoculate PDAplates in order to get spores for liquid culture inoculation. The liquidcultures in IC1+5 g/l K⁺Phtalate were grown 5 days at 27° C. (shaking180 rpm). Then, the cultures were centrifuged (5000 g, 10 min.). Fromthese samples, xylanase activity was measured by DNS Technique accordingto Miller et al. (ref. 18) TABLE G Active XYN2 production levels in C1(best producers) Active xylanase II concentration in Xylanase IIspecific culture media activityin culture media Untransformed UV18-253.9 U./ml 3.8 U./mg total prot. UV18-25::1064 clone 7-1 4.7 U./ml 4.7U./mg total prot. UV18-25::1064 clone 7-2 4.4 U./ml 4.3 U./mg totalprot. UV18-25::1065 clone 1-1 29.7 U./ml  25.6 U./mg total prot. UV18-25::1065 clone 1-2 30.8 U./ml  39.4 U./mg total prot. 

[0232] These data show that:

[0233] 1) Points 1 to 4 from example 2 are confirmed.

[0234] 2) C1 can be used as host for the secretion of heterologousfungal proteins.

[0235] (4) Summary

[0236] Table H shows the results for the plasmids with whichtransformation of UV18-25 was carried out. The Table shows expressionlevels for endoglucanase and cellobiohydrolase using heterologousexpression regulating sequences and signal sequences and also withhomologous expression regulating sequences and signal sequences. Thedetails of the various plasmids can be derived elsewhere in thedescription and from the figures. The production occurs at alkaline pHat a temperature of 35° C. TABLE H Expression data of transformedUV18-25 strain (% relative to parent UV18-25 strain) Total proteinsCMCase β-glucanase Culture mg/ml u/ml u/mg u/ml u/mg pH value UV18-25100%  100% 100% 100% 100% 7.90 1150-23 94% 105% 111% 140% 149% 7.90 -3096% 105% 110% 145% 151% 8.10 1152-3 94% 112% 120% 147% 156% 7.85 -4100%  105% 105% 132% 132% 7.90 1160-2 69%  81% 118%  90% 131% 7.90 -473%  72%  98%  83% 114% 8.35 -1 92%  95% 103% 120% 130% 8.45 1162-1102%  105% 103% 145% 142% 8.20 -11 112%  109%  98% 115% 103% 8.20 F6g-20104%  102%  98% 130% 125% 7.90 -25 — — — — —

[0237] E. Construction of an Aspergillus Sojae Gene Library

[0238] (1) Vector Library

[0239] Genomic DNA of A. sojae was isolated from protoplasts obtainedfrom ATCC 11906 using a previously described protocol (Punt, van denHondel, Methods Enzymol. 1992 216:447-457). After isolation DNA wasextracted from the protoplasts using the protocol described by Kolar etal., Gene 1988 62:127-34. Subsequently the DNA was partially digestedwith MboI to result in DNA fragments of an average size of 30-50 kb.

[0240] Vector pAOpyrGcosarp1, which was used for the construction of thegene library was constructed by ligation of a 3 kb BamHI-HindII fragmentfrom pANsCos1 (Osiewacz, Curr Genet. 1994 26:87-90) and a 3.2 kbAcc65I-HindIII fragment from pAO4.2 (De Ruiter-Jacobs et al., Curr.Genet. 1989 16:159-63) in Acc65I-BamHI digested pHELP1 (Gems et al.,Gene 1991 98:61-67). This cosmid vector carries the A. oryzae pyrGselection marker and is self-replicating in filamentous fungi.

[0241] MboI digested genomic DNA was ligated to BamHI-digestedpAOpyrGcosarp1, and the ligation mixture was packaged into phageparticles using the Stratagene Supercos1 vector kit (Stratagene Inc., LaJolla Calif.). This resulted in a total of ca. 30,000 individual clones,representing an approximate 30-fold representation of the A. sojaegenome. Stocks (in 15% glycerol) of pools of the resulting clones werestored at −80° C. for later use.

[0242] (2) High-Frequency Transformation

[0243] An A. sojae ATCC 11906 pyrG mutant was selected as a fluorooroticacid-resistant derivative from ATCC 11906, as described in WO 01/09352.This strain, A. sojae ATCC 11906pyrG, was transformed with two vectorscarrying the A. niger pyrG gene. One vector pAB4-1 (van Hartingsveldt etal., Mol. Gen. Genet. 206:71-75 (1987)) carries only the pyrG gene,whereas pAB4-arp1 (Verdoes et al., Gene 146:159-165 (1994)) carries thepyrG gene and the A. nidulans AMA1 sequence. Transformation of ATCC11906pyrG results in 5-10 transformants per microgram DNA from pAB4-1,whereas with pAB4-arp1 frequency were at least 10-100 fold higher.Phenotypic analysis of the transformants revealed that the pyrGphenotype of the pAB4-arp1 transformants was maintained only undercontinuous selection, whereas the pAB4-1 transformants were stable withand without selection for the pyrG phenotype. These results confirmautonomous replication of the introduced plasmid DNA in pAB4-arp1transformants. Similar results were obtained with alternative fungaltransformation vectors carrying the AMA1 sequence or derivativesthereof., e.g. pAOpyrGcosarp1.

[0244] (3) Construction of a Fungal Transformant Library

[0245]A. sojae ATCC11906pyrG or relevant mutants, in particular compactmorphology mutants thereof, was transformed with an A. sojae genelibrary based on transformation vector pAOpyrGcosarp1. This vectorresults in a high frequency of transformants with freely replicatingvector copies. Fungal protoplasts were treated as described in Punt andvan den Hondel, Methods Enzymol. 1992 216:447-457 with DNA from a cosmidlibrary carrying genomic fungal DNA clones from A. sojae orChrysosporium and serial dilutions of the transformed protoplasts wereplated on selective agar plates to determine the transformationfrequency obtained. The remaining protoplasts were regenerated inselective medium for a few hours and stored at 4° C. Based on theresults obtained for the transformation frequency (which depending ofthe experiment will reach values up to several thousand transformantsper microgram of cosmid library DNA), limiting dilutions of theregenerated protoplasts were plated in microtiter plates of 96, 248, oralternative well format, resulting in one transformed protoplast perwell. Plates were incubated at 35° C. to form fungal biomass. Theresulting transformant library is used for further experiments.

[0246] A similar strategy was used for the construction of a collectionof fungal transformants carrying mutant alleles of Chrysosporium CBH1.This strategy can also be used with a library of mutants derived fromany other gene of interest, whether generated by mutagenesis, geneshuffling or gene-evolution approaches.

[0247] F. Induction of Sproulation in Submerged Fermentation

[0248] Many fungi, such as Aspergillus sojae, do not show sporulationunder submerged fermentation. Here we describe a previously unknownapproach to obtain sporulation under these conditions. A. sojae ATCC11906 and in particular compact growth morphology mutants thereof weregrown in a synthetic growth medium supplemented with Yeast extract.Under these conditions rapid accumulation of biomass occurs in bothstatic and agitated cultures. However, no sporulation occurs in theculture fluid. A similar growth medium with the addition of 0.6 g/kgEDTA results in considerable yields of spores reaching up to 10⁹ sporesper ml culture fluid after incubation of 2-4 days at 35° C. SYNTHETICMEDIUM (+/− EDTA): g/kg medium KH₂PO₄  2.5 NH₄Cl  7.2 MgSO₄.7H₂0  0.7CaCl₂.2H₂0  0.2 Yeast Extract 20 ZnSO₄.7H₂0  0.015 CoCl₂.6H₂0  0.005CuSO₄.5H₂0  0.016 FeSO₄.7H₂0  0.040 H₃BO₄  0.005 KI  0.003 MnCl₂.2H20 0.012 Na₂MoO₄.2H20  0.003 EDTA (0.6 or 0.0) PH adjusted to 5.5 withNaOH/H₃PO₄

[0249] G. Transformation Systems for Chrysosporium and Aspergillus

[0250] (1) Cloning of the A. niger Orotate p-ribosyl Transferase GenepyrE

[0251] Numerous versatile transformation systems for filamentous fungiare based on the use of uridine-requiring mutant strains. These mutantstrains are either deficient in orotidine 5 phosphate decarboxylase(OMPD) or orotate p-ribosyl transferase (OPRT). (T. Goosen et al., CurrGenet. 1987, 11:499-503; J. Begueret et al., Gene. 1984 32:487-92.)Previously we have isolated the A. niger OMPD gene pyrG (W. vanHartingsveldt et al., Mol. Gen. Genet. 1987 206:71-5). The cloning ofthe A. niger OPRT gene (pyrE) was carried out by complementation of anA. niger FOA-resistant uridine-requiring non-pyrG mutant. Forcomplementation an A. niger cosmid library in vector pAOpyrGcosarp1 wasused. From the complementing transformants, genomic cosmid clones wereisolated, carrying the complementing A. niger gene, termed now pyrE. A5.5 kb SstII fragment carrying the pyrE gene was cloned in pBLUESCRIPT(™) (Stratagene) resulting in vector pBLUEpyrE. A 1.6 kb fragment ofthis vector spanning the pyrE coding region was sequenced to confirm thelocation of the OPRT gene (See FIG. 15).

[0252] (2) Auxotrophic Transformation System for Chrysosporiumlucknowense

[0253] Uridine-requiring Chrysosporium lucknowense strains were selectedas fluoroorotic acid resistant derivatives from C1 and UV18-25 bymethods described in PCT publication WO 01/09352. Selection offluoro-orotic acid resistant derivatives may result in the isolation oftwo types of uridine-requiring mutants, i.e. either orotidine 5phosphate decarboxylase (OMPD) mutants or orotate p-ribosyl transferase(OPRT)mutants (T. Goosen et al., Curr Genet. 1987, 11:499-503). Todetermine the nature of the Chrysosporium mutants obtained,transformation experiments were carried out with the available A. nigergenes pyrG (OMPD; vector pAB4-1, W. van Hartingsveldt et al., Mol. Gen.Genet. 1987 206:71-5) and pyrE (pBLUE-pyrE; OPRT). As shown in Table I,only transformation of the mutant strains with the pyrE gene resulted inprototrophic transformants, implying that the Chrysosporium strains areOPRT mutants. Following the Chrysosporium gene nomenclature we haveadopted, the mutants were designated pyr5. TABLE I Gene Source Vector¹⁻⁴UV18FOA^(R)#4 C1#B OMPD Aspergillus niger pAB4-1 − − (PyrG/pyr4)Aspergillus oryzae pAO4-2 − − Neurospora crassa pDJB3 − − OPRTAspergillus niger pBLUEpyrE + + (pyrE/pyr5)

[0254] (3) Construction and Use of Autonomously Replicating FungalTransformation Vectors.

[0255] Based on vector pBLUEpyrE two derivatives were generated carryingsequences providing autonomous replicative characteristics to thevectors when introduced in filamentous fungi. A 5.5 kb HindIII fragmentcarrying the Aspergillus nidulans AMA1 sequences (J. Verdoes et al.,Gene 1994 146:159-65) was introduced in the unique HindIII site ofpBLUEpyrE resulting in pBLUEpyrE-AMA. A 2.1 kb (partial) HindIIIfragment carrying human telomeric sequences (A. Aleksenko, L. Ivanova,Mol. Gen. Genet. 1998 260:159-64) was introduced in the unique HindIIIsite of pBLUEpyrE resulting in pBLUEpyrE-TEL. These vectors wereintroduced into Aspergillus and Chrysosporium OPRT mutant strainsresulting in prototrophic transformants. Several of the obtainedtransformants showed the ragged phenotype characteristic oftransformants carrying freely replicating plasmids (J. Verdoes et al.,Gene 1994 146:159-65).

[0256] (4) Transformation of Chrysosporium lucknowense

[0257] The protocol is based on a procedure originally used forAspergillus transformation (P. Punt, C. van den Hondel, Methods inEnzymology 1992 216:447-457). Rich medium (250 ml) was inoculated with10⁶ spores/ml of the pyr5 Chrysosporium mutant (supra) in a 1LErlenmeyer flask. The culture was grown for 24-48 hours at 35° C. in anair incubator (300 rpm). The mycelium was filtered through a sterileMiracloth(™) filter (Calbiochem) and washed with ca. 100 ml 1700 mosmolNaCl/CaCl₂ (0.27 M CaCl₂/0.6 M NaCl). The mycelium was weighed and thenkept on ice. Caylase(™) (Cayla) was added (20 mg per gram mycelium) and1700 mosmol NaCl/CaCl₂ (3.3 ml/g mycelium) and the suspension wasincubated in a 33° C. air incubator (100 rpm). The protoplasting wasfollowed under the microscope. After 1-3 hours of incubation, most ofthe mycelium was digested, leaving mostly protoplasts in the microscopicview of the preparation. The protoplasts were filtered through a sterileMyracloth filter and the filter was washed with 1 volume cold STC1700(1.2 M sorbitol/ 10 mM Tris.HCl pH 7.5/ 50 mM CaCl₂/35 mM NaCl). Theprotoplasts were spun down at 2500 rpm for 10 minutes at 4° C. Thepellet was resuspended in STC1700 and centrifuged again. Afterresuspending the pellet in STC1700, the protoplasts were counted.STC1700 was added to a final concentration of 2×10⁸ protoplasts per ml.

[0258] Vector DNA (pAB4-1 or pBLUE-pyrE, 1-10 μg) was pipetted into thebottom of a sterile tube and 1 μl 1M ATA (aurintricarbonic acid) and 100μl protoplasts (ca. 2×10⁷) were added to the DNA. A minus DNA negativecontrol was included in the experiment. After mixing, the protoplastswere incubated at room temperature for 25 minutes. PEG6000 (60% PEG/50mM CaCl₂/10 mM Tris pH 7.5) was added portionwise as follows: 250 μl,mix, 250 μl, mix, 850 μl and mix. The solution was kept at roomtemperature for 20 minutes. The tubes were then filled with 8 mlSTC1700, mixed and centrifuged at 2500 rpm for 10 minutes at 4 C., andthe pellet was suspended in 250 μl STC1700. Aloquots of the sample wereused for plating on selective medium. For pyr⁺ selection, plates wereprepared containing 1.5% Daishin agar, 1.2 M sorbitol, 1× AspA withnitrate, 2 mM MgSO₄.7 H₂O, 1× trace elements, 0.1% casaminoacids and 1%glucose. If selected for amdS (and pyr⁺), the plates contained 1.5%Oxoid agar, 1.2 M sorbitol, 2mM MgSO₄.7 H₂O, 1× trace elements, 1%glucose, 1× AspA without nitrate, 15 mM CsCl and 10 mM acetamide oracrylamide. The plates were incubated at 30 or35° C.

[0259] The spores and viable protoplasts before and after PEG6000treatment were counted by plating dilutions in STC1700 on minimal mediumplates with nitrate and with or without sorbitol. 100 μl of 10⁻¹, 10⁻²and 10⁻³ dilutions were plated on plates without sorbitol to count forspores and 100 μl of 10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵ dilutions were plated onplates with sorbitol to count the viable protoplasts.

[0260] Results of the transformations are shown in Table I.

[0261] H. Protein/Biomass Ratios

[0262] For Chrysosporium, Trichoderma, and Aspergillus strains producingcellulases or amylases, total dry solids were determined by passing ameasured aliquot of the whole broth through a preweighed filter, washingwith deionized water, and drying the cake and filter overnight at 60° C.and for one hour at 100° C. After cooling in a dessicator, biomass wasdetermined by subtracting the weight of the filter from the weight ofthe dry filter plus filter cake and dividing by the volume of brothremoved.

[0263] For Trichoderma and Aspergillus strains, the biomass was assumedto be equal to the total dry solids as there was little insolublematerial other than biomass at the time measurements were taken. ForChrysosporium strains producing cellulase, there was a significantquantity of cellulose in the medium, so biomass was determined as thedifference between total dry solids and cellulose. Cellulose was assayedas follows.

[0264] Measured aliquots of whole broth were centrifuged to removesolids and the supernatant was discarded. The pellet was resuspendedinto a volume of 0.1 N NaOH equal to the original broth volume and onetenth volume of 0.5 N NaOH was added. The mixture was incubated for fourhours at 65° C. This treatment dissolved everything except thecellulose. The alkaline mixture was cooled and centrifuged, and thesupernatant was discarded. The resulting pellet was washed twice byresuspension in deionized water and centrifugation. The washed pelletwas resuspended in deionized water, transferred to a preweighed pan anddried as described above. Cellulose concentration was determineddividing the dry weight by the volume of the aliquot assayed.

[0265] Protein was determined by the Bradford dye-binding procedure (M.Bradford, 1976, Anal. Biochem. 72:248) using an immunoglobulin standard.Protein/biomass ratios for selected expressed proteins in variousfilamentous fungal strains are presented in Table J. TABLE J g Proteinper Enzyme Strain g Biomass Neutral Cellulase Chrysosporium lucknowenseUV18-25 8.2 Neutral Cellulase Chrysosporium lucknowense UV26-2 6.0α-Amylase Aspergillus oryzae 108-318 0.89 Glucoamylase Aspergillus niger0.78 Glucoamylase Aspergillus niger 1.11 Acid Cellulase Trichodermareesei A-34 0.89 Acid Cellulase Trichoderma reesei A-1391 0.65 XylanaseTrichoderma reesei X-252 2.4

[0266] I. Expression and Secretion of Green Fuorescent Protein in A.sojae and C. lucknowense

[0267] As an example of a versatile and easily screenable reporterprotein, Green Fluorescent Protein (GFP) from the jellyfish Aequoriavictoria was expressed in A. sojae and C. lucknowense. Vectors carryingGFP (A. Santerre Henriksen et al., Microbiology. 1999, 145:729-734) andGlucoamylase-GFP fusion genes (pGPDGFP, C. Gordon et al., Microbiology.2000 146:415-26) were modified by replacing the glaa promoter with theconstitutively-expressed A. nidulans gpdA promoter. The vectors wereintroduced into A. sojae by cotransformation, using either the pyrG oramdS selection marker. Vector pGPDGFP and its derivatives wereintroduced in Chrysosporium by cotransformation using either the pyrE oramdS selection marker. Expression resulted in brightly fluorescent A.sojae and Chrysosporium transformants, confirming expression of GFP byboth vectors. Fluorescence of culture supernatants from transformantsexpressing Glucoamylase-GFP fusion protein indicated secretion of thefluorescently active fusion protein. Expression of fluorescent proteinwas also observed in spores (or spore-like propagules) obtained from thevarious transformants expressing the non-secreted cytoplasmic version ofthe fluorescent proteins.

[0268] J. Transfer of Fungal Growth Units

[0269] The wells of a 96-well microtiter plate are loaded with anappropriate medium, either manually with a multi-channel pipet or bymeans of an automated plate-handling system. A large volume increasesthe chance of cross-infection, whereas to avoid problems withevaporation the volume should not be too small. If using the COSTAR(™)3799 round-bottom plate, for example, 150 μl is an appropriate volume towork with. Plates are inoculated with spores from plate-grown coloniesusing toothpicks for transfer. Alternatively, plates can be inoculatedby pipetting small aliquots of suspensions of spores, protoplasts orhyphal elements. These suspensions may be derived from isolatedspore/protoplast solutions or from microplate grown sporulatingcultures. Inoculation can also be carried out from microtiter plateswith the use of a pin or a 96-pin tool.

[0270] Subsequently plates are incubated at 35° C. To minimizeevaporation, lidded plates may be employed, or the plates may be sealedwith a membrane that allows exchange of O₂, H₂O and CO₂ and sticks tothe surface of the plate. To further limit evaporation, acontrolled-atmosphere incubator may be used.

[0271] After three to four days of incubation, the amount of biomass isappropriate for efficient transfer to new microtiter plates containingfresh medium. For preparation of replica plates, a 96-pin tool is used.Daughter plates having different arrangements of the cultures may beprepared by manual or robotic pipetting or pin transfer. To ensure thepresence of transferable reproducing elements on the transfer pins, thepin tool is submerged into the microtiter plate culture and shaken for20 seconds. The pin tool is then carefully removed from the startingplate and a print is made into a new microtiter plate. A similarlyefficient transfer procedure can also be achieved by using amulti-channel pipet, transferring about 1 μl of the parent microtiterplate culture. In both cases efficient transfer is achieved due to thepresence of the transferable reproductive elements, such as spores,spore-like propagules, protoplasts, or hyphal or mycelial fragments.Protoplasts may be generated in the microplate wells by treatment withcell wall degrading enzymes and then transfer these protoplasts.Protoplast formation in microplates has been described by C. van Zeijlet al., J Biotechnol. 1997 59:221-224.

[0272] A further improvement of the transfer is obtained by incubatingthe microtiter plate cultures on a microtiter plate shaker at 35° C.This increases the number of transferable reproductive elements in thecultures. To store the microtiter plate cultures, glycerol is added to a15 % end concentration, and the plates are stored at −80° C. Forsubsequent transfer experiments plates are defrosted and transfer iscarried out as described before. Efficient transfer with wild-type orcommercial strains of A. niger and A. sojae was not feasible under theconditions used here, as these strains showed vigorous surface growthand aerial sporulation after one day. Aerial sporulation causes massivecross-contamination during transfer, and surface growth covering thewells subsequently precludes a large proportion of known assay methods.

[0273] K. Construction of a Fungal Expression Library for Gene Discovery

[0274] Based on the fungal expression vector pAN52-1NOT (EMBL accessionZ32524) or one of its derivatives, a vector was constructed in which aunique BamHI cloning site is present directly downstream of theconstitutively expressed broad fungal host range promoter for the A.nidulans gpdA gene (P. Punt et al., J. Biotechnol. 1991 17:19-33). Thisvector was constructed in such a way that genomic DNA fragments carryinga translation start codon (ATG) may be expressed. To provide a selectionmarker for this vector, a NotI-BamHI fragment from pBLUEpyrE was clonedin the NotI-Bg1II digested expression vector termed pAN52-BamHI,resulting in vector pAN52-pyrE. Chrysosporium genomic DNA fragments in asize range of 3-6 kb were obtained partial Sau3A digestion. Afterligation of these fragments into the BamHI-digested expression vectorpAN52-pyrE, a number of recombinant clones sufficient to cover the fullChrysosporium genome several times was obtained. A number of theseclones were pooled to cover at least 5-10 fungal genome equivalents.Plasmid DNA of these pools was prepared and used for transformation ofChrysosporium pyr5 or Aspergillus pyrE mutants. Transformant collectionswere generated in a microplate format as described above, and used forfurther functional/activity screening. Alternatively, an expressionlibrary may be constructed using specifically regulated Chrysosporiumpromoters, as described in PCT/NL99/00618.

REFERENCES CITED IN EXAMPLES

[0275] (The contents of the following, and all patents and referencescited hereinabove, are incorporated herein by reference):

[0276] 1. Calmels T. P., Martin F., Durand H., and Tiraby G. (1991)Proteolytic events in the processing of secreted proteins in fungi. J.Biotechnol. 17(1):51-66.

[0277] 2. Punt P. J., Dingemanse M. A., Jacobs-Meijsing B. J., PouwelsP. H., and van den Hondel C. A. (1988) Isolation and characterization ofthe glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillusnidulans. Gene 69(1):49-57.

[0278] 3. Shoemaker S., Schweickart V., Ladner M., Gelfand D., Kwok S.,Myambo K., and Innis M. (1983) Molecular cloning ofexo-cellobiohydrolase I derived from Trichoderma reesei strain L27.Bio/Technology October:691-696.

[0279] 4. Drocourt D., Calmels T., Reynes J. P., Baron M., and Tiraby G.(1990) Cassettes of the Streptoalloteichus hindustanus ble gene fortransformation of lower and higher eukaryotes to phleomycin resistance.Nucleic Acids Res. 18(13):4009.

[0280] 5. Mullaney E. J., Hamer J. E., Roberti K. A., Yelton M. M., andTimberlake W. E. (1985) Primary structure of the trpC gene fromAspergillus nidulans. Mol. Gen. Genet. 199(1):37-45.

[0281] 6. Yanisch-Perron C., Vieira J., and Messing J. (1987) ImprovedM13phage cloning vectors and host strains: nucleotide sequences of theM13mp18 and pUC9 vectors. Gene 33:103-119.

[0282] 7. Durand H., Baron M., Calmels T., and Tiraby G. (1988)Classical and molecular genetics applied to Trichoderma reesei for theselection of improved cellulolytic industrial strains, in Biochemistryand genetics of cellulose degradation, J. P. Aubert, Editor. AcademicPress. pp. 135-151.

[0283] 8. Lowry O. H., Rosebrough N. J., Farr A. L., and Randall R. J.(1951) Protein measurements with the folin phenol reagent. J. Biol. Chem193, 265-275.

[0284] 9. Parriche M., Bousson J. C., Baron M., and Tiraby G.Development of heterologous protein secretion systems in filamentousfungi. in 3rd European Conference on Fungal Genetics. 1996. Münster,Germany.

[0285] 10. Baron M., Tiraby G., Calmels T., Parriche M., and Durand H.(1992) Efficient secretion of human lysozyme fused to the Sh-blephleomycin resistance protein by the fungus Tolypocladium geodes. J.Biotechnol. 24(3):253-266.

[0286] 11. Jeenes D. J., Marczinke B., MacKenzie D. A., and Archer D. B.(1993) A truncated glucoamylase gene fusion for heterologous proteinsecretion from Aspergillus niger. FEMS Microbiol. Lett.107(2-3):267-271.

[0287] 12. Stone P. J., Makoff A. J., Parish J. H., and Radford A.(1993) Cloning and sequence-analysis of the glucoamylase gene ofneurospora-crassa. Current Genetics 24(3):205-211.

[0288] 13. Mörsky P. (1983) Turbidimetric determination of lysozyme withMicrococcus lysodeikticus cells: Reexamination of reaction conditions.Analytical Biochem. 128:77-85.

[0289] 14. Paluh J. L., Orbach M. J., Legerton T. L., and Yanofsky C.(1988) The cross-pathway control gene of Neurospora crassa, cpc-1,encodes a protein similar to GCN4 of yeast and the DNA-binding domain ofthe oncogene v-jun-encoded protein. Proc. Natl. Acad.

[0290] Sci. USA 85(11):3728-32.

[0291] 15. Nakari T., Onnela M. L., Ilmen M., Nevalainen K., andPenttilä M. (1994) Fungal promoters active in the presence of glucose,International patent application WO 94/04673

[0292] 16. Torronen A., Mach R. L., Messner R., Gonzalez R., KalkkinenN., Harkki A., and Kubicek C. P. (1992) The two major xylanases fromTrichoderma reesei: characterization of both enzymes and genes.Biotechnology 10(11):1461-5.

[0293] 17. Farkas V. (1985) Novel media for detection ofmicrobialproducers of cellulase and xylanase. FEMS Microbiol. Letters 28:137-140.

[0294] 18. Miller G. L. (1959) Use of dinitrosalicylic acid reagentfordetermination of reducing sugar. Anal. Chem. 31:426-428.

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1. A method of expressing a plurality of proteins encoded by a library of DNA vectors, wherein the library of vectors comprises a plurality of different vectors, each different vector comprising a different protein-encoding nucleic acid sequence, said nucleic acid sequence being operably linked to an expression-regulating region and optionally a secretion signal encoding sequence, the method comprising the steps of: (a) providing a filamentous fungus having a phenotype characterized by growth in suspension and characterized by the production of transferable reproductive elements in suspension; (b) stably transforming said filamentous fungus with said library of DNA vectors so as to introduce into each of a plurality of the individual fungi at least one heterologous protein-encoding nucleic acid sequence; (c) culturing the transformed mutant filamentous fungi under conditions conducive to formation of transferable reproductive elements in suspension; (d) separating from one another a plurality of transferable reproductive elements; and (e) culturing into monoclonal cultures or monoclonal colonies the individual transferable reproductive elements, under conditions conducive to expression of the heterologous proteins encoded by the heterologous protein-encoding nucleic acid sequences.
 2. A method of screening a plurality of proteins encoded by a library of DNA vectors for an activity or property of interest, comprising the steps of: (a) expressing the plurality of proteins in monoclonal filamentous fungal cultures or monoclonal filamentous fungal colonies, by the method of claim 1; and (b) screening individual tonal cultures or clonal colonies for the activity or property of interest.
 3. A method of producing a DNA molecule encoding a protein having an activity or property of interest, comprising the steps of: (a) expressing a plurality of proteins in monoclonal filamentous fungal cultures or monoclonal filamentous fungal colonies, by the method of claim 1; (b) screening individual clonal cultures or clonal colonies for the activity or property of interest; and (c) isolating DNA from a clonal culture or clonal colony exhibiting the activity or property of interest.
 4. A method of producing the nucleotide sequence of a DNA molecule encoding a protein having an activity or property of interest, comprising the steps of: (a) isolating DNA from a clonal culture or clonal colony exhibiting the activity or property of interest, by the method of claim 3; and (b) sequencing said DNA.
 5. A method of producing the amino acid sequence of a protein having an activity or property of interest, comprising the steps of: (a) producing the DNA sequence of the protein having an activity or property of interest, by the method of claim 4; and (b) converting said DNA sequence into an amino acid sequence.
 6. A method of screening a plurality of monoclonal filamentous fungal cultures or monoclonal filamentous fungal colonies for a metabolite having an activity or property of interest, comprising the steps of: (a) expressing a plurality of proteins in monoclonal filamentous fungal cultures or monoclonal filamentous fungal colonies, by the method of claim 1; and (b) screening each individual clonal culture or clonal colony for the activity or property of interest.
 7. A method of optimizing a protein's activity or property of interest, comprising the steps of: (a) providing a library of vectors which comprise DNA sequences encoding mutant forms of the protein; (b) providing a filamentous fungus having a phenotype characterized by growth in suspension and by the production of transferable reproductive elements in suspension; (c) stably transforming said filamentous fungus with said library of DNA vectors so as to introduce into each of a plurality of individual fungi at least one heterologous protein-encoding nucleic acid sequence; (d) culturing the transformed filamentous fungi under conditions conducive to the formation of transferable reproductive elements; (e) separating from one another a plurality of transferable reproductive elements; (f) culturing into clonal cultures or clonal colonies the individual transferable reproductive elements, under conditions conducive to expression of the heterologous proteins encoded by the heterologous protein-encoding nucleic acid sequences; (g) screening each individual organism, clonal culture, or clonal colony for an expressed protein having the activity or property of interest; (h) isolating one or more individual organisms, clonal cultures, or clonal colonies that express a protein exhibiting the activity or property of interest; (i) mutating the DNA from the isolated individual organisms, clonal cultures, or clonal colonies that encodes the protein exhibiting the activity or property of interest; (j) providing a library of vectors which comprise the mutated DNA sequences obtained in step (i); and (k) repeating steps (b) through (g), until the property or activity of interest either reaches a desirable level or no longer improves.
 8. The method of claim 7, further comprising between steps (h) and (i) the steps of: culturing one or more of the individual organisms, clonal cultures, or clonal colonies isolated in step (h); isolating the expressed protein exhibiting the activity or property of interest; and evaluating the isolated protein for the property of interest.
 9. The method of claim 2, wherein the screening step is carried out by high-thoughput screening.
 10. The method of claim 3, wherein the screening step is carried out by high-thoughput screening.
 11. The method of claim 4, wherein the screening step is carried out by high-thoughput screening.
 12. The method of claim 5, wherein the screening step is carried out by high-thoughput screening.
 13. The method of claim 6, wherein the screening step is carried out by high-thoughput screening.
 14. The method of claim 7, wherein the screening step is carried out by high-thoughput screening.
 15. The method of claim 8, wherein the screening step is carried out by high-thoughput screening.
 16. The method of any one of claims 1-15, wherein the fungus has a phenotype characterized by a culture viscosity, when cultured in suspension, of less than 200 cP at the end of fermentation when grown with adequate nutrients under optimal or near-optimal conditions.
 17. The method of any one of claims 1-15, wherein the fungus has a phenotype characterized by a culture viscosity, when cultured in suspension, of less than 100 cP at the end of fermentation when grown with adequate nutrients under optimal or near-optimal conditions.
 18. The method of any one of claims 1-15, wherein the fungus has a phenotype characterized by culture viscosity, when cultured in suspension, of less than 60 cP at the end of fermentation when grown with adequate nutrients under optimal or near-optimal conditions.
 19. The method of any one of claims 1-15, wherein the fungus has a phenotype characterized by a culture viscosity, when cultured in suspension, of less than 10 cP at the end of fermentation when grown with adequate nutrients under optimal or near-optimal conditions.
 20. The method of any one of claims 1-15, wherein the vectors comprise a fungal signal sequence.
 21. The method of claim 20, wherein the fungal signal sequence is the signal sequence of a fungal gene encoding a protein selected from the group consisting of cellulase, β-galactosidase, xylanase, pectinase, esterase, protease, amylase, polygalacturonase and hydrophobin.
 22. The method of any one of claims 1-15, wherein the vectors comprise a nucleotide sequence encoding a selectable marker.
 23. The method of any one of claims 1-15, wherein the vectors comprise an expression-regulating region region operably linked to the protein-encoding nucleic acid sequence.
 24. The method of claim 23, wherein the expression regulating region comprises is an inducible promoter.
 25. The method of any one of claims 1-15, wherein the fungus is of the class Euascomycetes.
 26. The method of claim 25 wherein the fungus is of the order Onygenales.
 27. The method of claim 25 wherein the fungus is of the order Eurotiales.
 28. The method of any one of claims 1-15, wherein the fungus is of the division Ascomycota, with the proviso that it is not of the order Saccharomycetales.
 29. The method of any one of claims 1-15, wherein the fungus is of a genus selected from the group consisting of: Aspergillus, Trichoderma, Chrysosporium, Neurospora, Rhizomucor, Hansenula, Humicola, Mucor, Tolypocladium, Fusarium, Penicillium, Talaromyces, Emericella and Hypocrea.
 30. The method of claim 29 wherein the fungus is of a genus selected from the group consisting of Aspergillus, Fusarium, Chrysosporium, and Trichoderma.
 31. The method of claim 30, wherein the fungus is Chrysosporium strain UV18-25 having accession number VKM F-3631 D.
 32. The method of claim 30, wherein the fungus is Trichoderma longibrachiatum strain X-252.
 33. The method of claim 30, wherein the fungus is Aspergillus sojae strain pclA.
 34. The method of claim 30, wherein the fungus is Aspergillus niger strain pclA.
 35. The method of any of claims 1-15, wherein the expressed protein to biomass ratio is at least 1:1.
 36. The method of claim 35, wherein the expressed protein to biomass ratio is at least 2:1.
 37. The method of claim 36, wherein the expressed protein to biomass ratio is at least 6:1.
 38. The method of claim 37, wherein the expressed protein to biomass ratio is at least 8:1.
 39. The method of any of claims 1-15, wherein the transferable reproductive elements are individual fungal cells.
 40. The method of any of claims 1-15, wherein the transferable reproductive elements are spores.
 41. The method of any of claims 1-15, wherein the transferable reproductive elements are hyphal fragments.
 42. The method of any of claims 1 -15, wherein the transferable reproductive elements are micropellets.
 43. The method of any of claims 1 -15, wherein the transferable reproductive elements are protoplasts.
 44. A method for obtaining a protein having an activity or property of interest, comprising the steps of: (a) screening a plurality of proteins encoded by a library of DNA vectors for an activity or property of interest, by the method of claim 2; (b) culturing on appropriate scale the monoclonal culture or monoclonal colony expressing the activity or property of interest, under conditions conducive to expression of the heterologous proteins encoded by the heterologous protein-encoding nucleic acid sequences; and (c) isolating the expressed protein.
 45. A method for obtaining a protein having an activity or property of interest, comprising optimizing the activity or property of interest by the method of claim 7 or claim 8, culturing on an appropriate scale an individual organism, clonal culture, or clonal colony isolated in the final step (h), and isolating the expressed protein from the culture.
 46. A method of making a library of transformed filamentous fungi, comprising the steps of: (a) providing a filamentous fungus having a phenotype characterized by growth in suspension and characterized by the production of transferable reproductive elements in suspension; and (b) stably transforming said filamentous fungus with a library of DNA vectors so as to introduce into each of a plurality of the individual fungi at least one heterologous protein-encoding nucleic acid sequence; wherein the library of DNA vectors comprises a plurality of different vectors, each different vector comprising a different protein-encoding nucleic acid sequence, said nucleic acid sequence being operably linked to an expression-regulating region and optionally a secretion signal encoding sequence.
 47. A library of transformed filamentous fungi, prepared by the method of claim
 43. 48. A method for obtaining a transformed filamentous fungal host expressing a protein having an activity or property of interest, comprising the steps of: (a) screening a plurality of proteins encoded by a library of DNA vectors for an activity or property of interest, by the method of claim 2; and (b) isolating the monoclonal culture or monoclonal colony expressing the activity or property of interest. 